Methods for purifying pertussis toxin and peptides useful therefor

ABSTRACT

The present invention relates to reagents and methods for purifying pertussis toxin (PT).

FIELD OF THE INVENTION

The present invention relates to reagents and methods for purifyingpertussis toxin (PT).

BACKGROUND OF THE INVENTION

Pertussis toxin (PT) is produced by Bordetella pertussis is a maincomponent in all vaccines against whooping cough. PT is typicallycombined with tetanus and diphtheria toxoids. Industrial production ofPT is typically achieved by cultivating B. pertussis in defined media PTis then isolated from the supernatant and purified by using thewell-known techniques (i.e., U.S. Pat. Nos. 6,399,076; 5,877,298; and,Sekura, et al. J. Biol. Chem. 258:14647-14651, 1983; Bogdan, et al.Appl. Env. Micro. 69 (10): 6272-6279, October 2003). The majority ofknown methods each require the use of matrix-bound bovine fetuin (BF) orasialofetuin, the source and purity of which is critical. The use ofbovine-derived reagents has led to some concern over bovine-relateddiseases such as bovine spongioform encephalopathy (BSE).

Those of skill in the art have therefore desired a method for purifyingPT that does not rely on BF. One such method is described by Bogdan, etal. (Appl. Env. Micro. 69 (10): 6272-6279, October 2003) Peptides havingthe ability to mimic the glycosidic moiety of bovine fetuin by bindingto PT were identified using a phage display system. Three peptides (3G5:NGSFSGF; 3G8: NGSFSGC; and, 3G2: DGSFSGF) having the consensus sequenceXGSFSGX (X is any amino acid) were identified as having PT-bindingcapacity. 3G2 was also utilized in an affinity column to purify PT froma partially purified PT preparation.

Additional methods for designing and utilizing peptides to purify PT inthe absence of bovine products are desired by those of skill in the art.Provided herein are reagents and methodologies for affinity purificationof PT without the use of fetuin in any form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A) Schematic representation of the gurmarin library. Positionsof the library that are translated to an amino acid sequence arehighlighted. The sequence of the protein portion (59 amino acids inlength) is shown in the single letter amino acid code, where Xrepresents any amino acid. Portions of the library that are nottranslated are indicated as gray boxes. (a) T7-promoter for optimal invitro transcription of library, (b) TMV—Tabaco Mosaic Virus translationinitiation sequence for perfect in vitro translation of library, (c)His₆-tag for efficient affinity purification of PROfusion™ library, (d)structural, flexible linker, (e) gurmarin core with two randomized loopscontaining 5 and 9 amino acids respectively, (f) structural, flexiblelinker and (g) optimized linker for efficient coupling withpuromycin-acceptor-molecule. B) The construction of the gurmarinPROfusion™ library is a multi-step process comprising the followingreactions: PCR, in vitro transcription, chemical ligation of RNA withpuromycin-oligonucleotide linker, in vitro translation, oligo-dTpurification, reverse transcription and His-tag purification.

FIG. 2. Schematic representation of a PROfusion™ selection cycle.

FIG. 3. Selected gurmarin variants that should be tested for bindingactivity towards PT. Conserved sequence motifs are highlighted bycolored boxes.

FIG. 4. Sequence analysis of the gurmarin selection round 4 against PT.The amino acid sequence of individual variants is shown in the singleletter amino acid code. Constant, flanking regions of the library andconstant regions of the gurmarin scaffold are highlighted. The positionof the randomized loops 1 and 2 are indicated.

FIG. 5. Sequence analysis of the gurmarin selection round 5 a against PT(epoxy). The amino acid sequence of individual variants is shown in thesingle letter amino acid code. Constant, flanking regions of the libraryand constant regions of the gurmarin scaffold are highlighted. Theposition of the randomized loops 1 and 2 are indicated.

FIG. 6. Sequence analysis of the gurmarin selection round 5 b against PT(strep). The amino acid sequence of individual variants is shown in thesingle letter amino acid code. Constant, flanking regions of the libraryand constant regions of the gurmarin scaffold are highlighted. Theposition of the randomized loops 1 and 2 are indicated.

FIG. 7. Sequence analysis of the gurmarin selection round 6 a against PT(strep). The amino acid sequence of individual variants is shown in thesingle letter amino acid code. Constant, flanking regions of the libraryand constant regions of the gurmarin scaffold are highlighted. Theposition of the randomized loops 1 and 2 are indicated.

FIG. 8. Sequence analysis of the gurmarin selection round 6 b against PT(strep). The ammo acid sequence of individual variants is shown in thesingle letter amino acid code. Constant, flanking regions of the libraryand constant regions of the gurmarin scaffold are highlighted. Theposition of the randomized loops 1 and 2 are indicated.

FIG. 9. Selected PP26 variants that will be tested for binding activitytowards PT. Conserved sequence motifs are highlighted.

FIG. 10. Sequence analysis of the PP26 selection round 4 against PT. Theamino acid sequence of individual variants is shown in the single letteramino acid code. Constant, flanking regions of the library arehighlighted.

FIG. 11. Sequence analysis of the PP26 selection round 5 a against PT(epoxy). The amino acid sequence of individual variants is shown in thesingle letter amino acid code. Constant, flanking regions of the libraryare indicated by light yellow boxes. Conserved sequence motifs arehighlighted.

FIG. 12. Sequence analysis of the PP26 selection round 5 b against PT(strep). The amino acid sequence of individual variants is shown in thesingle letter amino acid code. Constant, flanking regions of the libraryare indicated by light yellow boxes. Conserved sequence motifs arehighlighted.

FIG. 13. Sequence analysis of the PP26 selection round 6 a against PT.The amino acid sequence of individual variants is shown in the singleletter amino acid code. Constant, flanking regions of the library areindicated by light yellow boxes. Conserved sequence motifs arehighlighted.

FIG. 14. Sequence analysis of the PP26 selection round 6 b against PT.The amino acid sequence of individual variants is shown in the singleletter amino acid code. Constant, flanking regions of the library arehighlighted.

FIG. 15. Immobilization of synthetic biotinylated core peptides toStreptavidin sepharose and verification of binding to purified PT. Theunbound fraction of PT was analyzed by separation of 1/40 volume of thesupernatant after binding on a 12% NuPage gel with MES running-buffer(upper gel). To analyze sepharose bound PT 50% of the eluate wasseparated on 12% NuPage gel with MES running-buffer (lower gel).Detection was performed by silver staining. Defined amounts of purifiedPT were used as standard for quantification, except for the gurmarinpeptides 15 and 9.

FIG. 16. Purification of PT out of Sample A (left gel) and Sample B(right gel). To analyze sepharose bound PT 50% of the eluate wasseparated on 12% NuPage gel with MES running-buffer (lower gel).Detection was performed by silver staining. Defined amounts of purifiedPT were used as standard for quantification, except for the gurmarinpeptide 9.

FIG. 17. Optimization of the washing conditions of bound PT out ofsample A or B to immobilized peptides pp26 clone 9 and 15 and gurmarinclone 9 and 15 using 3 washes of 50 mM Tris/HCl, pH 7.5 or 50 mMacetate, pH 6. The PT were analyzed on 12% Bis Tris gels and visualizedby silver staining. PPM: protein perfect marker.

FIG. 18. Optimization of washing conditions of bound PT out of sample Bto immobilized peptides pp26 clone 9 using 3 to 20 washes of 50 mMTris/HCl, pH 7.5 or 50 mM acetate, pH 6. The PT was analyzed on 12% BisTris gels and visualized by silver staining.

FIG. 19. Elution of PT from peptide streptavidin sepharose with 0.2 to2.0 MMgCl₂ in 50 mM Tris/HCl. Peptide bound PT was displaced from thepeptide-streptavidin sepharose by three consecutive washes with theindicated elution buffers (20 μl each). Remaining material wassubsequently eluted with gel loading buffer. All elutions were analyzedon 12% Bis Tris gels (1×MES running buffer) and visualized by silverstaining.

FIG. 20. Elution of PT from peptide streptavidin sepharose under acidic(50 mM glycine, pH 2.5) or basic (100 mM carbonat buffer, pH 10.5)conditions. Peptide bound PT was displaced from the peptide streptavidinsepharose (20 μl containing ˜200 pmol of one peptide) by threeconsecutive washes with with the indicated elution buffers (40 μl each).Remaining material was subsequently eluted with gel loading buffer. Allelutions were analyzed on 12% Bis Tris gels (1×MES running buffer) andvisualized by silver staining. 1/40 volume of the flow through afterpeptide streptavidin sepharose incubation with sample A was analyzed wasanalyzed on the same gel for each peptide.

FIG. 21. Small scale column purification of PT from sample B onstreptavidin sepharose with immobilized pp26 peptide 9 as affinityligand (A) an gel estimation of the yield of purified PT (B).

FIG. 22. Small scale column purification of PT from sample B onstreptavidin sepharose with immobilized gurmarin peptide 15 as affinityligand (A) an gel estimation of the yield of purified PT (B).

FIG. 23. PT binding to peptide streptavidin sepharose in dependence ofvarying amounts of peptide (as indicated) used for immobilization onstreptavidin sepharose (per 1 ml). Amount of bound PT was quantified bydirect comparison to defined amounts of purified PT on the same gel. Asan example, pp26/9 is plotted against the amount of peptide used forimmobilization per ml of streptavidin sepharose. Maximal binding wasestimated at approximately 100-150 pmol PT.

FIG. 24. PT yield as function of varying amounts of input material(sample B) per μl peptide streptavidin sepharose or 6.85 μl asialofetuinsepharose. The amount of eluted PT was calculated on the basis of directcomparison to defined amounts of purified PT on the same gel and listedin the Table 12.

FIG. 25. Reutilization of peptide sepharose for repeated PT binding andelution. Bound PT to streptavidin sepharose were 4 times eluted with 100mM Carbonate buffer at pH 10.5 and the column matrix was regeneratedwith 10 mM HCl.

FIG. 26. PT elution fractions after FPLC-column purification on pp26/9peptide streptavidin sepharose (0.5 ml) from sample B. The elutionfractions (0.5 μl of each 500 μl elution) were analyzed by PAGE (12%Bis-Tris-Gel, MES running buffer) and silver staining. Defined amountsof purified PT were separated on the same gel for direct comparison.Concentration of PT was determined by measuring the absorbance of theelution fractions at 280 nm (A₂₈₀) and compared to purified PT standards(see table).

SUMMARY OF THE INVENTION

The present invention relates to methods for purifying pertussis toxin(PT). In one embodiment, a method for generating a DNA-protein fusion bycovalently bonding a nucleic acid reverse-transcription primer bound toa peptide acceptor to an RNA, translating the RNA to produce a peptideproduct such that the protein product is covalently bound to the primer,reverse transcribing the RNA to produce a DNA-protein fusion, andtesting the fusion product to identify those containing PT bindingpeptides. The sequence of the peptide is then identified by sequencing.In other embodiments, peptides are provided that have PT-bindingcapacity and are useful for purifying PT from complex biological fluids.Also provided are peptides bound to solid supports and/orchromatographic media for use in purifying PT from complex biologicalfluids and methods for carrying out such purifications.

DETAILED DESCRIPTION

The present invention provides reagents and methodologies for a newmethod for purifying pertussis toxin (PT). As described above, one suchmethod has been demonstrated by Bogdan, et al. In that method, phagedisplay was utilized to identify PT-binding peptides. For the purposesof practicing the present invention, PT includes naturally expressed PT,detoxified PT (genetically or otherwise), natural or other PT variants,recombinant PT, PT fragments, or other versions of PT (see, for example,U.S. Pat. Nos. 6,399,076; 6,168,928; 6,018,022; 5,977,304; 5,965,385;5,856,122; 5,877,298; 5,433,945; 5,358,868; 5,332,583; 5,244,657;5,221,618; 5,085,862; 4,997,915). In most cases, chemical detoxificationis performed following purification of PT. Any form of PT is suitablefor use in practicing the present invention as long as a reagent asdescribed herein has the ability to bind the particular form of PT.Within this application, all cited references, patents, and patentapplications are incorporated herein by reference.

The present invention also relates to the use of recombinant technologyto identify PT-binding peptides. The present invention providesadvantages over methods already known in the art. In addition, novelpeptides useful in purifying PT are provided herein. In one embodiment,a method for generating a DNA-protein fusion by covalently bonding anucleic acid reverse-transcription primer bound to a peptide acceptor toan RNA, translating the RNA to produce a peptide product such that theprotein product is covalently bound to the primer, reverse transcribingthe RNA to produce a DNA-protein fusion, and testing the fusion productto identify those containing PT binding peptides. The sequence of thepeptide is then identified by sequencing. In certain embodiments, theRNA moiety may be removed from the complex by treatment with anRNA-degrading compound such as RNase H. Photocrosslinking reagents andpeptide acceptors are also useful in practicing the present invention.This system and related reagents have been described elsewhere in, forexample, U.S. Pat. No. 6,416,950 (Lohse, et al); U.S. Pat. No. 6,429,300(Kurz, et al.); U.S. Pat. No. 6,436,665 (Kuimelis, et al.); U.S. Pat.No. 6,602,685 (Lohse, et al); and, U.S. Pat. No. 6,623,926 (Lohse, etal).

In practicing the invention, a reagent such as a nucleic acid, peptide,fusion, ligand, affinity complex, or the like may be non-diffusivelybound or attached to a solid support. In order to be non-diffusivelybound or attached, the reagent is chemically or physically combined withthe solid support such that the reagent does not move in the presence ofliquid from a region of high concentration of reagent to a region of lowconcentration of reagent. A solid support is any column (i.e., unpackedor packed chromatographic media, column material), bead, test tube,microtiter dish, solid particle (i.e., agarose or sepharose), microchip(i.e., silicon, silicon-glass, or gold chip), membrane (i.e., themembrane of a liposome or vesicle), or other medium to which a reagentmay be bound or attached, either directly or indirectly (for example,through other binding partner intermediates such as an antibody, ProteinA, Protein G, streptavidin, biotin).

In preferred embodiments, the reagent is a substance or compound havingthe ability to bind PT. More preferably, the reagent is a substance orcompound having the ability to reversibly bind PT. Even more preferably,the reagent is a peptide having the ability to at least bind, andpreferably reversibly bind PT within a liquid containing componentsother than PT. A reagent that reversibly binds PT is one that binds PTunder certain conditions (adsorption), and releases PT under otherconditions (desorption). For example, the reagent may bind PT whenexposed to conditions of neutral pH and release PT following exposure toconditions of acidic or basic pH. Thus, the ability of the reagent tobind PT (i.e., the equilibrium dissociation constant or K_(d)) may bemanipulated by altering the conditions under which the reagent is incontact with PT. Other conditions may also be changed, includingtemperature, ionic strength (i.e., concentration of an ionic salt suchas sodium chloride or magnesium chloride, for example), solventconcentration, presence or absence of a competitor reagent/freeligand/analogue, polar properties, among others as is known in the art.

In certain embodiments, an affinity matrix (i.e., a PT-binding peptidebound to a solid support) is utilized to separate a desired component(i.e., PT) from a complex mixture found within a liquid, biological orotherwise. In certain cases, it may be desirable to purify PT from acomplex biological fluid such as a bacterial lysate or other compositionin which PT does not comprise the majority of components within thefluid (as determined by SDS-PAGE, for example). In other cases, PT maybe isolated from a composition that has been partially purified for PTsuch that the majority of the components within the fluid is representedby PT (a composition consisting of approximately greater than or equalto 50% PT). For example, a composition in which PT consists of about 50%or more of the total protein in the composition as determined bySDS-PAGE would under most circumstances be considered partiallypurified.

To purify PT, a composition containing PT may be μlaced into contactwith a PT-binding reagent, preferably a reversibly binding PT-bindingreagent, that is bound to a solid support for a sufficient period oftime such that PT and the PT-binding reagent bind to one another to forma complex. Non-PT components are then washed away. One or moreconditions (i.e., pH) are then changed such that the K_(d) of the PT-PTbinding reagent bond increases, and PT is released from the complex.Released PT is then collected and prepared for further use. Such aseparation may be termed affinity purification and products so purifiedreferred to as being affinity purified.

Chromatographic techniques that are generally considered by those ofskill in the art to be less selective than affinity purificationtechniques may also be used in practicing the present invention. As isknown in the art, such techniques may include, for example,size-exclusion chromatography, ion-exchange chromatography,reverse-phase chromatography, and hydrophobic-interactionchromatography. Any of these techniques (including affinitypurification) may be carried out using the proper solid support in a lowpressure chromatography (LPC), high pressure liquid chromatography(HPLC), or fast protein liquid chromatography (FPLC) setting, forexample. Suitable solid supports and equipment for carrying out suchtechniques are widely available in the art. In practicing the presentinvention, both affinity chromatography and the more generalizedtechniques may be combined as needed to either partially purify astarting material (i.e., complex biological fluid such as a bacteriallysate), purify material, or further purify affinity- orotherwise-purified material (i.e., affinity purified PT).

Peptides have been identified that bind PT and are described herein.Certain peptides have been found to bind PT with high affinity. Suchpreferred PT binding peptides include:

RSSHCRHRNCHTITRGNMRIETPNNIRKDA (pp26-5); STMNTNRMDIQRLMTNHVKRDSSPGSIDA(pp26-6); RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9);RSWRDTRKLHMRHYEPLAIDSYWDHTLRDA (pp26-15);SGCVKKDELCARWDLVCCEPLECIYTSELYATCG (G-9);SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG (G-10);SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15); and,SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG (G-19).Of these, especially preferred peptides include:

RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9); and,SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15).

Further contemplated are related peptides such as, for example,fragments, variants orthologs, homologues, and derivatives, for example,that possess at least one characteristic or activity (i.e., activity,antigenicity) of the peptide. A fragment comprises a truncation of thesequence (i.e., nucleic acid or polypeptide) at the amino terminus (withor without a leader sequence) and/or the carboxy terminus of thepeptide. Fragments may also include variants, orthologs, homologues, andother variants having one or more amino acid additions or substitutionsor internal deletions as compared to the parental sequence. In preferredembodiments, truncations and/or deletions comprise about one amino acid,two amino acids, five amino acids, 10 amino acids, 20 amino acids, 30amino acids, 40 amino acids, 50 amino acids, or more. A variant is asequence having one or more sequence substitutions, deletions, and/oradditions as compared to the parental sequence. Variants may benaturally occurring or artificially constructed. Such variants may beprepared from the corresponding nucleic acid molecules. In preferredembodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30,or from 1 to 40, or from 1 to 50, or more than 50 amino acidsubstitutions, insertions, additions and/or deletions.

Substitutions may be conservative, or non-conservative, or anycombination thereof. Conservative amino acid modifications to thesequence of a polypeptide (and the corresponding modifications to theencoding nucleotides) may produce polypeptides having functional andchemical characteristics similar to those of a parental polypeptide. Forexample, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a non-native residuesuch that there is little or no effect on the size, polarity, charge,hydrophobicity, or hydrophilicity of the amino acid residue at thatposition and, in particular, does not result in decreasedimmunogenicity. Suitable conservative amino acid substitutions are shownin Table I.

TABLE I Original Preferred Residues Exemplary SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln GlnAsp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala AlaHis Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine LeuLeu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyricAcid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr LeuPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

A component such as PT may be said to be purified when it has beenseparated from at least about 50% of the proteins, lipids,carbohydrates, or other materials with which it is originally found(i.e., a bacterial lysate). It is preferred that the component beseparated from at least about 95-100%, 90-95%, 80-90%, 70-80%, 60-70% or50-60% of the total protein content of a composition as determined bySDS-PAGE, for example. In certain embodiments, a purified component isone that is useful in inducing an immune response in a host to whom thecomponent has been administered, either alone or in combination withother agents. The immune response may include the production ofantibodies that bind to at least one epitope of PT or Bordetellapertussis, for example, and/or the generation of a cellular immuneresponse against cells expressing PT. The response may be an enhancementof a current immune response by, for example, causing increased antibodyproduction, production of antibodies with increased affinity for theantigen, or an increased cellular response (i.e., increased T cells).Other measures of an immune response are known in the art and would besuitable in determining whether or not an immune response has occurred.

PT isolated using the methods described herein may be prepared aspharmaceutical compositions. Preferred pharmaceutical compositionsinclude, for example, PT in a liquid preparations such as a suspensions,syrups, or elixirs. Preferred injectable preparations include, forexample, peptides suitable for parental, subcutaneous, intradermal,intramuscular or intravenous administration such as sterile suspensionsor emulsions. For example, PT may be prepared as a composition inadmixture with a suitable carrier, diluent, or excipient such as sterilewater, physiological saline, glucose or the like. The composition mayalso be provided in lyophilized form for reconstituting, for instance,in isotonic aqueous, saline buffer. Such compositions may also beprepared and utilized as a vaccine as described in, for example, U.S.Pat. Nos. 5,877,298 and 6,399,076 (Vose, et al.) as well asInternational App. No. PCT/CA96/00278. PT prepared as indicated hereinmay also be combined with other antigens from disease-causing organismssuch as Corynbacterium (i.e., diphtheria), Clostridium (i.e., tetanus),polio virus (i.e., IPV, OPV), hepatitis virus, Neisseria (i.e.,meningitis), Streptococcus, Hemophilus, or other pertussis antigens(i.e., filamentous hemagglutinin, pertactin, and agglutinogens), amongothers as is known in the art.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Materials And Methods A. Pertussis Toxin (PT)

PT is a heterooligomeric protein complex with a MWr of 109 kD (consistsof the 6 subunits S1, S2, S3, 2 x S4, S5). A high purity (>99.99%)preparation, formulated as an ammonium sulfate precipitate, wasutilized. A PT-specific ligand (asialofetuin) recognizing the nativehexameric complex was also utilized. Asialofetuin is available in asolubilized and in sepharose immobilized form.

B. Gurmarin Library Selection

Gurmarin is a 35-residue polypeptide from the Asclepiad vine Gymneasylvestre. It has been utilized as a pharmacological tool in the studyof sweet-taste transduction because of its ability to selectivelyinhibit the neural response to sweet tastants in rats. It has noapparent effect in humans. It has been suggested that thetaste-suppressing of gurmarin might be due to the peptide either bybinding directly to a sweet-taste receptor or interacting with adownstream target in the sweet-taste-transduction system.

Gurmarin belongs to the family of “knottins”, a group of structurallyrelated proteins, typically less than 40 residues in length. Knottinsbind to a diverse range of molecular targets that includes proteins,sugars and lipids but share a common scaffold comprising a smalltriple-stranded antiparallel β-sheet and disulphide bound framework.

A specialized gurmarin-library was designed with 15 randomized aminoacid positions, as shown below:

Wild-type gumarin: qqCVKKDELCIPYYLDCCEPLECKKVNWWDHKCig Gumarin core:CVKKDELCXXXXXXCCEPLECXXXXXXXXXCWithin the gurmarin core sequence, X represents any amino acid. Thislibrary was validated to yield high affinity binders against proteintargets. The gurmarin library combines a set of advantages that makes itthe best choice for a selection against the PT-toxin for at least thefollowing reasons: limited flexibility makes up for high entropic costin conforming to target topology; theoretically fewer amino acids forhigher affinities than in linear libraries; resistant to proteases; andsusceptibility to redox-elution conditions in downstream applications.The gurmarin library was constructed using process shown in FIG. 1.

1. PCR of Starting Oligonucleotides

Three gel-purified oligos were used to construct the gurmarin librarywith two randomized loops. 1 nmole of gurmarin template (≈ca. 6·10¹⁴sequences) 5′-AGT GGC TCA AGC TCA GGA TCA GGC TGC GTC AAG AAA GAC GAGCTC TGC NNS NNS NNS NNS NNS NNS TGC TGT GAG CCC CTC GAG TGC NNS NNS NNSNNS NNS NNS NNS NNS NNS TGC GGC AGC GGC AGT TCT GGG TCT AGC-3′, wasamplified for 6 rounds of PCR (94° C., 1 min; 65° C., 1 min; 72° C., 1min) using 1 μM of the 5′-His-Tag Primer 5′-TAA TAC GAC TCA CTA TAG GGACAA TTA CTA TTT ACA ATT ACA ATG CAC CAT CAC CAT CAC CAT AGT GGC TCA AGCTCA GGA TCA-3′ and 1 μM of the 3′-Primer 5′-TTT TAA ATA GCG GAT GCT ACTAGG CTA GAC CCA GAA CTG CCG CT-3′ using Taq-polymerase and analyzed on a2% agarose gel, which indicated a representative library had beenconstructed.

2. In Vitro Transcription

dsDNA was transcribed into RNA using the RiboMax Express In vitrotranscription kit from Promega. After incubation for 45 min at 37° C.,DNase I was added and the incubation at 37° C. continued for anadditional 15 minutes. This mixture was subjected to a phenol/chloroformextraction. Excess of NTPs was removed by NAP-5 gel filtration(Pharmacia). RNA was analyzed on a 6%-TBU-gel, and indicated that thedsDNA had been efficiently transcribed.

3. Chemical Coupling of RNA And Puromycin-Oligonucleotide Linker

Purified RNA will be annealed (85° C., 1 min cool down to 25° C. at aramp of 0.3° C./s) to a 1.5-fold excess of puromycin-oligonucleotidelinker PEG2A18: 5′-psoralen-UAG CGG AUG C A₁₈ (PEG-9)₂ CC puromycin(nucleotides shown in italics represent 2′-O-methyl-derivatives). Thecovalent coupling is performed by illumination for 15 min at RT (RT)with UV-light (365 nm). The reaction product was analyzed on 6%-TBU geland indicated the linking reaction had proceeded efficiently.

4. In Vitro Translation

Ligated RNA was translated using the rabbit reticulocyte lysate fromPromega in the presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). Aftera 30 min incubation at 30° C., KCl and MgCl₂ were added to a finalconcentration of 530 mM and 150 mM respectively and a sample wasanalyzed on 4-20% Tris/glycine-SDS-PAGE. The gel indicated that thetranslation reaction was successful.

5. Oligo-dT Purification

Molecules (mRNA-protein fusions) were isolated by incubation with oligodT magnetic beads (Miltenyi) in incubation buffer (100 mM Tris-HCl pH8.0, 10 mM EDTA, 1 mM NaCl and 0.25% Triton X-100) for 5 min at 4° C.PROfusion™ molecules were isolated by filtration throughMiniMACS-columns (Miltenyi), washing with incubation buffer and elutionwith water. A sample was analyzed on 4-20% Tris/glycine-SDS-PAGE, andindicated that the reaction was successful.

6. Reverse Transcription

A corresponding cDNA strand was generated by reverse transcription withSuperScript II Reverse Transcriptase (Gibco BRL) under the manufacture'srecommended conditions using a 5-fold excess of 3′-Primer. A sample wasanalyzed on 4-20% Tris/glycine-SDS-PAGE, and indicated that the reactionwas successful.

7. His-Tag Purification

Reverse transcribed PROfusion™ molecules were mixed with Ni-NTA-agarose(50 μl/10 pmole PROfusion™) (QIAGEN) in HBS buffer (20 mM HEPES pH 7.0,150 mM NaCl, 0.025% Triton X-100, 100 μg/ml sheared salmon sperm DNA, 1mg/ml BSA) and incubated for 60 min at RT under gentle shaking. Ni-NTAwas then filtrated, washed with HBS/5 mM imidazole and PROfusions™ wereeluted with HBS/150 mM imidazole. A sample was analyzed on 4-20%Tris/glycine-SDS-PAGE, and indicated that the purification wassuccessful. 20 pmole (≈ca. 1·10¹³ sequences) of PROfusion™ moleculeswill be used as input for each selection.

B. Linear Peptide Library PP26 For Selection

A specialized linear peptide library PP26 with 26 randomized amino acidpositions was also designed using the following construct:T7-TMV-MGRGS-HHHHHH-ARS-XXXXXXXXXXXXXXXXXXXXXXXXXX-DANAPK-ASAI Thesequence of the protein portion (50 amino acids in length) is shown inthe single letter amino acid code, where X represents any amino acid.Portions of the library that are not translated include: (a) T7: theT7-promoter for optimal in vitro transcription of library; and, (b) TMV:the Tabaco Mosaic Virus translation initiation sequence for perfect invitro translation of library. MGRGS represents a structural, flexiblelinker. HHHHHH represents a His₆-tag for efficient affinity purificationof PROfusion™ library. ARS represents a second structural, flexiblelinker. DANAPK represents a third structural, flexible linker. ASAIrepresents an optimized linker for efficient coupling withpuromycin-acceptor-molecule.

This library was validated to yield high affinity binders againstprotein targets. The PP26 library combines two major advantages thatmakes it an excellent choice for the selection of chromatographicaffinity reagents: high flexibility: can conform to the topology of thetarget; and robustness due to the absence of a conserved structure theresulting binders are resistant to harsh biophysical conditions

1. PCR of Starting Oligonucleotides

Three gel-purified oligos were used to construct the gurmarin librarywith two randomized loops. 1 nmole of PP26 template (≈ca. 6·10¹⁴sequences) 5′-AGC GGA TGC CTT CGG AGC GTT AGC GTC SNN SNN SNN SNN SNNSNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNN SNNSNN SNN SNN AGA TCT AGC ATG ATG ATG ATG A-3′, was amplified for 6 roundsof PCR (94° C., 1 min; 65° C., 1 min; 72° C., 1 min) using 1 μM of the5′-His-Tag Primer 5′-TAA TAC GAC TCA TAG GGA CAA TTA CTA TTT ACA ATT ACAATG GGA CGT GGC TCA CAT CAT CAT CAT CAT CAT GCT AGA TCT -3′ and 1 μM ofthe 3′-Primer 5′-AA TTA AAT AGC GGA TGC CTT CGG AGC GTT AGC -3′ usingTaq-polymerase and confirmed by analysis on a 2% agarose gel.

2. In Vitro Transcription

dsDNA was transcribed into RNA using the RiboMax Express In vitrotranscription kit from Promega. After incubation for 45 min at 37° C.,DNase I was added and the incubation at 37° C. continued for anadditional 15 minutes. This mixture was subjected to a phenol/chloroformextraction. Excess of NTPs was removed by NAP-5 gel filtration(Pharmacia). Transcription of RNA was confirmed by analysis on a6%-TBU-gel.

3. Chemical Coupling of RNA and Puromycin-Oligonucleotide Linker

Purified RNA will be annealed (85° C., 1 min

cool down to 25° C. at a ramp of 0.3° C./s) to a 1.5-fold excess ofpuromycin-oligonucleotide linker PEG2A18: 5′-psoralen-UAG CGG AUG C A₁₈(PEG-9)₂ CC puromycin (nucleotides shown in italics represent2′-O-methyl-derivatives). The covalent coupling is performed byillumination for 15 min at RT (RT) with UV-light (365 nm). The reactionwas confirmed by analysis of the reaction product on 6%-TBU gel.

4. In Vitro Translation

Ligated RNA was translated using the rabbit reticulocyte lysate fromPromega in the presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). Aftera 30 min incubation at 30° C., KCl and MgCl₂ were added to a finalconcentration of 530 mM and 150 mM respectively and translationconfirmed by analysis on 4-20% Tris/glycine-SDS-PAGE.

5. Oligo-dT Purification

Molecules (mRNA-protein fusions) were isolated by incubation with oligodT magnetic beads (Miltenyi) in incubation buffer (100 mM Tris-HCl pH8.0, 10 mM EDTA, 1 mM NaCl and 0.25% Triton X-100) for 5 min at 4° C.PROfusion™ molecules were isolated by filtration throughMiniMACS-columns (Miltenyi), washing with incubation buffer and elutionwith water. A sample was analyzed to confirm the reaction on 4-20%Tris/glycine-SDS-PAGE.

6. Reverse Transcription

A corresponding cDNA strand was generated by reverse transcription withSuperScript II Reverse Transcriptase (Gibco BRL) under the manufacture'srecommended conditions using a 5-fold excess of 3′-Primer. A sample wasanalyzed to confirm transcription on 4-20% Tris/glycine-SDS-PAGE.

7. His-Tag Purification

Reverse transcribed PROfusion™ molecules were mixed with Ni-NTA-agarose(50 μl/10 pmole PROfusion™ (QIAGEN) in HBS buffer (20 mM HEPES pH 7.0,150 mM NaCl, 0.025% Triton X-100, 100 μg/ml sheared salmon sperm DNA, 1mg/ml BSA) and incubated for 60 min at RT under gentle shaking. Ni-NTAwas then filtrated, washed with HBS/5 mM imidazole and PROfusions™ wereeluted with HBS/150 mM imidazole. A sample was analyzed to confirm thereaction on 4-20% Tris/glycine-SDS-PAGE. 20 pmole (≈ca. 1·10¹³sequences) of PROfusion™ molecules will be used as input for eachselection.

C. Target Preparation

In the PROfusion™ technology highly diverse substance libraries, whichare composed of up to 10¹³ different PROfusion™ molecules (mRNA-Proteinfusions), are selected against a wanted target (protein, sugar or lipid)for high affinity binding. In this process the targets will typically beimmobilized to solid phases. These solid phase are preferentiallymagnetic beads that allow fast and efficient handling during theselection process and give low background.

1. Test Targets for Nuclease Activity

Targets—5 μg PRP and 0.5 μg PT—were contacted with 0.12 pmoleradioactive labeled PROfusion™ library molecules at 4° C. and RT (RT)followed by an incubation for 1 h and 16 h respectively. The integrityof PROfusion™ molecules after incubation was confirmed by 4-20%Tris/glycine SDS-PAGE and subsequent autoradiography. Degradation ofPROfusion™ molecules was not detected, thus demonstrating that thetargets are free of nucleases.

2. Test Targets for Protease Activity

Targets—5 μg PRP and 0.5 μg PT—were contacted with 1 μg purifiedGST-protein at 4° C. and RT followed by an incubation for 1 h and 16 hrespectively. The integrity of GST-protein after incubation was analyzedby 4-20% Tris/glycine SDS-PAGE and subsequent Coomassie Brilliant Bluestaining. Degradation of GST-protein was not detected, thusdemonstrating that the targets are free of proteases.

D. Immobilization of PT 1. Reconstitution of PT

500 μl of the precipitate (2.26 mg/ml) as delivered by Aventis Pasteurwere centrifuged at 21.400×g for 45 min at RT. The supernatant wasdiscarded; the pellet was dissolved in 1100 μl CTW-buffer (0.286 gNaHCO₃, 0.170 g Na₂CO₃, 50 μl Tween-80, add to 50 ml MilliQ H₂O). Tocheck the quality of this PT preparation a dilution series (250 ng, 500ng, 1 μg, 2.5 pg, 5 μg and 15 μg) was separated on a 4-12% BisTrisSDS-PAGE, run in MES-buffer). At least 4 bands could be clearlyseparated, corresponding to the subunits S1 (28 kD), S2 (23 kD), S3 (22kD) and S4 (11.7 kD). The smallest protein S5 (9.3 kD) in the PT-complexcould not be seen. Probably, this band co-migrates in this gel systemwith the only slightly larger S4 subunit.

2. Coupling Strategy

Several methods were established for immobilization of proteins tomagnetic particles. In principle two major strategies are used: primaryamino groups and sulfhydryl groups of the target protein are tetheredcovalently to epoxy-activated magnetic beads (Dynal) forming stabileamide or thioether bounds. This reaction is performed in the presence ofammonium sulfate to promote the reaction and typically results in a veryefficient coupling of the target protein. Anyhow, certain proteins seemto undergo structural changes under these conditions resulting in abound but not native and/or inactive conformation; and, primary aminogroups and sulfhydryl groups of the target protein are tetheredcovalently to NHS-ester activated biotin derivatives (Pierce)subsequently followed by an immobilization of now biotinylated proteinto streptavidin magnetic beads (Dynal)

Typically, covalent coupling of a target protein to epoxy beads ispreferred if reaction conditions are suitable for a given target sincethis method guarantees that only the target is presented on the beads.In the case of a biotin/streptavidin coupling the beads also presentstreptavidin that could lead to the enrichment of anti-streptavidinbinder during a selection. Therefore, Phylos has established specializedmethods to preclear PROfusion™ libraries for streptavidin binders to gethigh quality results for a given target. But in total a covalentcoupling typically results in a faster enrichment of target specificbinders. In the specific case of PT it is most reasonable to start witha covalent coupling strategy since it is known that ammonium sulfateincubation does not influence the functionality of the PT-protein.

3. Optimization of Coupling Conditions to Epoxy Beads (Dynal)

The coupling conditions for PT were optimized in several independentexperiments (different ammonium sulfate concentrations (0.5-2.0 M) anddifferent beads/target-ratios were applied, as well as time- andtemperature dependency (2 min-16 h; 8° C.-RT). Best results wereobserved for the following reaction condition: A final volume of 300 μl,consisting of 100 μg PT, 3.3·10⁸ beads and a final ammonium sulfateconcentration of 1M was incubated in a time course for 2 min to 60 minat RT in a 2 ml Eppendorf tube. After incubation the tube was was placedin a magnet for 4 min to pull down the beads and the supernatant wasstored for subsequent gel analysis. The beads were washed once with 1 mlHEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X100) andan aliquot of beads (5% of the beads) were analyzed on a 4-12% BisTrisSDS-PAGE to determine the amount of associated protein. It was foundthat coupling of PT to epoxy beads occurs very efficiently even afteronly a two minute reaction.

4. Semi-Preparative Coupling of PT to Epoxy Beads

2.6 mg dry epoxy-activated beads (M-270, Dynal) (˜1.7-10⁸ beads) wereresuspended in 1 ml phosphate buffer (19 mM NaH₂PO₄, 81 mM Na₂HPO₄, pH7.4) and equilibrated for 10 min. The equilibration was repeated twotimes with fresh phosphate buffer. Subsequently the beads were directlyused in a coupling reaction with 480 pmole reconstituted PT (1 μg/μl inCTW buffer) in 1 M ammonium sulfate (final volume 157 μl). Afterincubation at RT for 15 min under continues agitation the beads werewashed with 300 μl HBS-buffer, followed by three washing steps withHEPES-buffer and finally resuspended in 240 μl HEPES-buffer and storedin aliquots at 4° C. The effectiveness of the coupling reaction waschecked by a SDS-polyacrylamidgel-analysis of all wash fractions, thesupernatant of the coupling reaction and the fraction of PT which wasremovable from the washed beads by SDS-loading-buffer.

5. Analysis of Epoxy-Bead Immobilized PT for Its Binding to Asialofetuin

40 μl of the PT-derivatized beads were incubated with 320 pmoleasialofetuin in HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025%Triton-X100) for 1 h at RT (final reaction volume 200 μl), washed 2-7times with 200 μl HEPES-buffer and finally resuspended in 30 μLHEPES-buffer. 50% of the beads were analyzed on SDS-PAGE to confirm thereaction.

Tests of these PT derivatized beads after one week of storage at 4° C.showed a reduced asialofetuin binding capacity indicating that thematerial looses its performance by long term storage. Thus,PT-derivatized beds have to be prepared fresh and quality controlled foreach selection round. Since this procedure is quite time consuming, analternative immobilization strategy involving a biotinylation of PT wasevaluated.

6. Semi-Preparative Biotinylation of PT

A biotinylation reaction was performed by incubation of 0.4 mg (˜3.65nmole) reconstituted PT (1 μg/μl in CTW buffer) with 25 μgEZ-link-sulfo-NHS-LC-LC-biotin (PIERCE) in a final volume of 740 μl 50mM HEPES, 150 mM NaCl, 0.2% Triton-X100. After an incubation period of 2h on ice under permanent agitation the biotinylation reaction wasquenched by addition of 74 μl 1M Tris/HCl pH 7.0. Subsequently, theprotein was dialyzed against HEPES-buffer (20 mM HEPES pH 7.0, 150 mMNaCl, 0.025% Triton X100) at 4° C. using a Slide-a-lyzer cassette(PIERCE, 3500 MWCO 0.5-3 ml) to remove the excess of biotinylationreagent. The biotinylated PT was removed from the dialysis cassette andstored in aliquots at −20° C.

7. Quality Control of Biotinylated PT Using a BIAcore Instrument

The quality of the biotinylation reaction was controlled by analysis ofthe interaction of biotinylated PT with a BIAcore streptavidin chipusing BIAcore instrument (BIAcore 2000). It was also possible to detectthe binding of asialofetuin to chip immobilized biotinylated PT (bindingsignal of ˜400 RU to immobilized PT; unspecific binding of ˜100 RU tothe control cell).

F. Analysis of Biotinylated PT for Binding to Streptavidin MagneticBeads and to Asialofetuin 1. Binding of Biotinylated PT to StreptavidinMagnetic Beads

20 μl streptavidin magnetic beads (Dynal) were incubated with 20 pmoleof biotinylated PT in 1×HBS-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 1mg/ml BSA, 100 μg/ml salmon sperm DNA, 0.025% Triton-X100) for 1 h atRT, washed 3×with HEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025 %Triton X100) and resuspended in 16 μl SDS-gel-loading buffer. 8 μl wereanalyzed by SDS-PAGE to confirm conjugation. In a negative controlexperiment under comparable conditions, free PT (not biotinylated) didnot interact with streptavidin magnetic beads.

2. Binding of Asialofetuin to Bead Immobilized Biotinylated PT

20 μl streptavidin magnetic beads (Dynal) were incubated with 20 pmoleof biotinylated PT in 1×HBS-buffer for 1 h at RT, washed 4× withHEPES-buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X100).Subsequently, beads with immobilized biotinylated PT were incubated with40 pmole asialofetuin in HEPES-buffer for 1 h at RT. After 4 washes withHEPES-buffer beads were resuspended in 16 μl SDS-gel-loading buffer. 8μl were analyzed by SDS-PAGE to confirm binding. A simultaneousincubation of biotinylated PT and asialofetuin to the streptavidinmagnetic beads instead of serial incubations resulted as well in bindingof asialofetuin to biotinylated PT. In a comparable control experiment,it was determined that asialofetuin did not interact with thestreptavidin magnetic beads non-specifically. Similar quality controlswith biotinylated PT that has been stored for one week at −20° C. showedno significant decrease in streptavidin and/or asialofetuin bindingcompetence. Therefore, biotinylated PT was used as standard target insubsequent selections.

Example 2 Isolation of Peptides Selective for PT

The gurmarin PROfusion™ library and PT immobilized to magnetic beadswere then contacted under strictly controlled stringency conditions.These conditions allow predominately those variants of the PROfusion™library showing elevated affinity for PT to bind to the targets. Afterextensive washes that dilute unwanted, non-specific binding variants,the bound PROfusion™-molecules are eluted from the beads and aresubjected to a new PROfusion™-formation cycle as shown in (FIG. 2). Bysuccessive rounds of selection and re-amplification along with a fineadaptation of stringency conditions a population of highly specificbinding molecules to the given target is enriched (10). Subsequently theDNA-portion of this population is cloned into an E. coli plasmid vectorto isolate individual variants that can be analyzed in detail bysequencing.

Six successive selection rounds against immobilized PT have beenperformed with the gurmarin PROfusion™-library. According to theperception described above, biotinylated PT immobilized to streptavidinbeads has been used in these selections (Table 1). In selection round 4,a low background binding of the gurmarin pool to streptavidin beads hasbeen observed which might indicate a starting enrichment of bead and/orstreptavidin binding gurmarin variants. Therefore, in the followingfifth selection round two individual selections were performed usingbiotin/streptavidin immobilized PT as target and epoxy bead coupled PT,respectively. In both selections, a clear background correctedenrichment of target binding was observed (Table 1). This trend has beenconfirmed in the sixth selection round using biotin/streptavidinimmobilized PT, clearly indicating an accumulation of PT-bindingvariants (Table 1).

A. Cloning of Selected Gurmarin Binder Pools

The gurmarin DNA-pools resulting from selection rounds R4, R5 and R6were cloned into the pCR® 2.1-TOPO®-vector using the TOPO TA Cloning®kit (Invitrogen). The gurmarin DNA was ligated to the pCR®2.1-TOPO®-vector in different concentrations. For 6 μl reactions, 0.5μl, 2 μl and 4 μl of the gurmarin pool DNA were used respectively. Theligation was performed according to the manufacturer's instructions.

Two (2) μl of these ligations were transformed into 20 μl of the E. coliTop 10 F′ competent cells (Invitrogen) and spread out on LB platescontaining 50 μg/ml Kanamycin and 0.5% Glucose. From each of thesetransformations 150 single colonies were picked to a masterplatecontaining 50 μg/ml Kanamycin and 0.5% Glucose to repress T7 dependentprotein expression and a second plate containing X-Gal and IPTG for ablue white screening. For each transformation, 96 of the colonies fromthe repressed masterplate corresponding to the white colonies from theblue white test were used to inoculate a 96 well LB agar plate and 500μl liquid cultures (LB containing 50 μg/ml Kanamycin and 0.5% Glucose).The 96 well agar plates were sent out for commercial sequencing service.The liquid cultures were mixed with 500 μl 40% Glycerol, frozen inliquid nitrogen and stored at −80° C.

From each individual clone, plasmid DNA was prepared and subjected to anautomated DNA-sequencing procedure using a M13-primer 5′-TGT AAA ACG ACGGCC AGT-3′. As shown in FIGS. 3-8, a single gurmarin sequence variantbegins to be significantly enriched in selection round 4 andrepresents >90% of all sequences after selection round 6. This clearlyindicates that this variant probably binds with the highest affinity toPT. In addition to this most prominent sequence variant, a variety ofother gurmarin sequences have been enriched that partially share commonsequence motifs. This finding indicates that these other sequences showaffinity towards PT as well.

B. PP26 Affinity Selection Against Immobilized PT

In parallel to the gurmarin selection six successive selection roundsagainst immobilized PT have been performed with the PP26PROfusion™-library. Biotinylated PT immobilized to streptavidin beadshas been used in these selections (Table 2). In selection round 4, a lowbackground binding of the gurmarin pool to streptavidin beads has beenobserved which might indicate a starting enrichment of bead and/orstreptavidin binding PP26 variants. Therefore, in the following fifthselection round two individual selections were performed using on theone hand biotin/streptavidin immobilized PT as target and on the otherhand epoxy bead coupled PT. In both selections a clear backgroundcorrected enrichment of target binding have been detected (Table 2).This trend was confirmed in the sixth selection round usingbiotin/streptavidin immobilized PT, thus, clearly indicating anaccumulation of PT-binding variants.

C. Cloning of Selected PP26 Binder Pools

The PP26 DNA-pools resulting from selection rounds R4, R5 and R6 werecloned into the pCR® 2.1-TOPO®-vector using the TOPO TA Cloning® kit(Invitrogen). The PP26 DNA was ligated to the pCR® 2.1-TOPO®-vector indifferent concentrations. For 6 μl reactions 0.5 μl/2 μl and 4 μl of thegurmarin pool DNA were used respectively. The ligation was performedaccording to the manufacturer's instructions. 2 μl of these ligationswere transformed into 20 μl of the E. coli Top 10 F′ competent cells(Invitrogen) and spread out on LB plates containing 50 μg/ml Kanamycinand 0.5% Glucose. From each of these transformations 150 single colonieswere picked to a masterplate containing 50 μg/ml Kanamycin and 0.5%Glucose to repress T7 dependant protein expression and a second platecontaining X-Gal and IPTG for a blue white screening. For eachTransformation 96 of the colonies from the repressed masterplatecorresponding to the white colonies from the blue white test were usedto inoculate a 96 well LB agar plate and 500 μl liquid cultures (LBcontaining 50 μg/ml Kanamycin and 0.5% Glucose). The 96 well agar plateswere sent out for commercial sequencing service. The liquid cultureswere mixed with 500 μl 40% Glycerol, frozen in liquid nitrogen andstored at −80° C.

D. Sequencing of Individual Binder Variants

From each individual clone plasmid DNA was prepared and subjected to anautomated DNA-sequencing procedure using a M13-primer 5′-TGT AAA ACG ACGGCC AGT-3′. As shown in FIGS. 9-14, two main variants have been enrichedduring the selection rounds. Both variants share a common conservedsequence motif This finding indicates that the side chains of theconserved amino acids putatively establish a direct interaction with acertain PT surface region. Furthermore, at least 4 additional variantshave been enriched at lesser extent. Since these variants do notcomprise the above mentioned conserved sequence motif it can beconcluded that these variants potentially bind to different surfaceregions of PT.

E. Validation of Selected PT-Binding Gurmarin- and PP26-Variants

Since the selections were performed withPROfusion™-molecules—mRNA-peptide-fusions—it is necessary in the firststep of the post selection analysis to check the free peptides for theirability to bind do the target. In the next step, those variants thatestablish their target binding through the peptide and not the nucleicacid portion are subjected to a specificity test in the presence of APprocess fluids. By this measure, those variants should be identifiedthat are most suitable to the AP process.

1. Test of Free Peptides for Their Binding Capacity to PT

For a qualitative binding assay of free peptides of single enrichedgurmarin- and PP26-binder variants the TNT T7 coupled Reticolocyte LysatSystem (Promega #L5540) was used, as follows. DNA of single bindercandidates was amplified by colony-PCR out of the glycerol stock ofbinder clones. To avoid mutations during PCR a proofreading polymerase(Pwo) was used. The PCR products were analyzed on a 2% agarose gel. 5.0μl of PCR product were used as template for coupled in vitrotranscription/translation reaction using the TNT system in a finalvolume of 53 μl according to the manufacturers instructions. Expressedbinder candidates were subsequently purified by Ni-NTA chelatchromatography (QIAGEN). Radioactively labeled His-tag purified bindercandidates (˜40-70 fmol of each peptide) were incubated withbiotinylated PT immobilized on streptavidin-magnetic beads for 1 h atRT. The beads were washed 3× with HBS-buffer and then resuspended inwater and analyzed by liquid scintillation counting. In controlexperiments each candidate was incubated with streptavidin beads only(without PT). The best binder candidates of PP26 and gurmarin wereidentified (Tables 3 and 4, below) and were subjected to the followingspecificity test.

2. Specificity Test of Gurmarin and PP26 Variants in the Presence ofProcess Fluids

For a semi-quantitative binding and specificity assay of free gurmarinand PP26 peptides in the presence of Aventis Pasteur process fluids thepeptides were first produced as PROfusion™, purified to homogeneity andthan transferred to free peptides by an S1-nuclease digest. Foramplification of a sufficient amount of DNA of the selected bindervariants (10 Gurmarin clones and 7 PP26 clones) a PCR was performedusing a PCR product from TNT expression as template. After 10 cycles ofPCR (94° C., 30 sec; 60° C., 30 sec; 72° C., 30 sec) the samples wereanalyzed on a 2% agarose gel. dsDNA (PCR product) was transcribed intoRNA using the RiboMax Express In vitro transcription kit from Promega.After incubation for 45 min at 37° C., DNase I was added and theincubation at 37° C. continued for an additional 15 minutes. Thismixture was subjected to a phenol/chloroform extraction. Excess of NTPswas removed by NAP-5 gel filtration (Pharmacia). RNA was analyzed on a6%-TBU-gel.

Purified RNA was annealed (85° C., 1 min cool down to 25° C. at a rampof 0.3° C./s) to a 1.5-fold excess of puromycin-oligonucleotide linkerPEG2A18: 5′-psoralen-UAG CGG AUG C A₁₈ (PEG-₉)₂ CC puromycin(nucleotides shown in italics represent 2′-O-methyl-derivatives). Thecovalent coupling was performed by illumination for 15 min at RT (RT)with UV-light (365 nm). The reaction product was analyzed on 6%-TBU gel.Ligated RNA was translated using the rabbit reticulocyte lysate fromPromega in the presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). Aftera 30 min incubation at 30° C., KCl and MgCl₂ were added to a finalconcentration of 530-mM and 150 mM respectively and a sample wasanalyzed on 4-20% Tris/glycine-SDS-PAGE. mRNA-protein fusions(PROfusions™) were isolated by incubation with oligo dT magnetic beads(Miltenyi) in incubation buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 1mM NaCl and 0.25% Triton X-100) for 5 min at 4° C. PROfusion™ moleculeswere isolated by filtration through MiniMACS-columns (Miltenyi), washingwith incubation buffer and elution with water. A sample was analyzed on4-20% Tris/glycine-SDS-PAGE.

To remove the MRNA part of the mRNA-protein fusions the oligo dTpurified molecules were digested with S1-Nuclease (S1-Nuclease cleavesthe DNA-part of the Puromycin linker) according to the manufacturersinstructions. Samples of the PROfusion molecules before and afterS1-digest were analyzed on 4-12% Bis/Tris SDS-PAGE. Streptavidin beads(M280 Dynal) were washed in HBS and incubated o/n at 4° C. BiotinylatedPT (900 pmol) was incubated with 900 μl Strepbeads (preblocked in HBSbuffer) for 1 h at RT. After immobilization of PT, the beads wereblocked with biotin (2 mM biotin in HBS) for 1 min and immediatelywashed 4× with HBS buffer to remove any traces of biotin. Control beads(without PT) were blocked with biotin in the same way.

For binding analysis of the selected peptides several parallel reactionswere set up, as follows: negative control only with biotin blockedStreptavidin beads; positive control with PT immobilized on Streptavidinbeads; background control with biotin blocked beads in combination with¼ volume Aventis Pasteur sample-solution C (flow through 1. AF column);mix of PT in combination with ¼ volume of sample-solution C; backgroundcontrol with biotin blocked beads in combination with ¼ volume ofAventis Pasteur sample-solution E (culture medium); mix of PT incombination with 1 volume of sample-solution E; reactions 3-6 wereperformed to investigate the capacity of the selected peptides to bindPT specifically in the presence of samples provided by Aventis Pasteur.Binding was done for 1 h at RT in the presence of a protease inhibitormix (complete mini™ ROCHE), to avoid degradation of the peptides. Afterwashing with HBS solution the beads were analyzed by scintillationcounting.

As shown in Table 3, three (#9, 10 19) of the ten tested gurmarinvariants show a target binding to PT that is not influenced by any ofthe AP process fluids. These variants are the most promising candidatesfor affinity chromatographic applications within the AP process.

As shown in Table 4 three (#5, 6 9) of the seven tested PP26 variantsshow target binding to PT that is not reduced by the AP process fluids.These variants are the most promising candidates for further affinitychromatographic applications within the AP process.

TABLE 3 Post selection analysis of gurmarin-variants* # seq # peptidesequence test 1 test 2 1 194227MHHHHHHSGSSSGSGCVKKDELCAGSVGHCCEPLECLRRFLNLRWCGSGSSGSS — n.d. 2 194238MHHHHHHSGSSSGSGCVKKDELCIVMRAPCCEPLECLRRYMLKHMCGSGSSGSS — n.d. 3 194239MHHHHHHSGSSSGSGCVKKDELCKAFRYSCCEPLECLRKWLKARFCGSGSSGSS — n.d. 4 194251MHHHHHHSGSSSGSGCVKKDELCLRSSIDCCEPLECLYKWMQRRLCGSGSSGSS — n.d. 5 194210MHHHHHHSGSSSGSGCVKKDELCWPRRHKCCEPLECLLEMLERKRCGSGSSGSS — n.d. 6 194261MHHHHHHSGSSSGSGCVKKDELCMSMACVCCEPLECKYHGYFWLCGSGSSGSS — n.d. 7 194214MHHHHHHSGSSSGSGCVKKDELCAVWFDVCCEPLECTYQSGYYWLCGSGSSGSS — n.d. 8 194226MHHHHHHSGSSSGSGCVKKDELCEPWYWRCCEPLECVYTSGYYYSCGSGSSGSS — n.d. 9 194259MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECIYTSELYATCGSGSSGSS ✓ ✓ 12 194297MHHHHHHSGSSSGSGCVKKDELCVFYFPNCCEPLECRWVNDNYGWCGSGSSGSS ✓ — 13 194330MHHHHHHSGSSSGSGCVKKDELCMSMACVCCEPLECKYHGYFWLCGSGSSGSS ✓ — 14 194479MHHHHHHSGSSSGSGCVKKDELCTTASKSCCEPLECKWTNEHFGTCGSGSSGSS ✓ — 15 194511MHHHHHHSGSSSGSGCVKKDELCSQSVPMCCEPLECKWFNENYGICGSGSSGSS ✓ — 16 194533MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECIYTSELYATCGSGSSGSS ✓ — 17 194486MHHHHHHSGSSSGSGCVKKDELCARWDLVCCEPLECLGHGLGYAYCGSGSSGSS — n.d. 18 194668MHHHHHHSGSSSGSGCVKKDELCMWSREVCCEPLECYYTGWYWACGSGSSGSS — — 10 194264MHHHHHHSGSSSGSGCVKKDELCELAVDECCEPLECFQMGHGFKRCGSGSSGSS ✓ ✓ 19 194737MHHHHHHSGSSSGSGCVKKDELCELAVDECCEPLECTKGDLGFRKCGSGSSGSS ✓ ✓ 20 194716MHHHHHHSGSSSGSGCVKKDELCELAIDVCCEPLECLGHGLGYAYCGSGSSGSS ✓ n.d. 21 194720MHHHHHHSGSSSGSGCVKKDELCELAIDVCCEPLECLGHGLGYAYCGSGSSGSS — — 11 194328MHHHHHHSGSSSGSGCVKKDELCNWVTPMRCEPLECLGHGLGYAYCGSGSSGSS ✓ n.d. *Test 1represents the target binding ability of free peptides (0) and test 2represents the binding specificity of variants in the presence of APprocess fluids (0). Variants that are positive in both assays are 9, 10,and 19.

TABLE 4 Post selection analysis of PP26-variants* # seq # peptidesequence test 1 test 2 1 197569MGRGSHHHHHHARSDWELSPPHVAITTRHLINCTDGPLLRDANAPKASAI — n.d. 2 197536MGRGSHHHHHHARSLNGESTSNILTTSRKVTEWTGYTASVDANAPKASAI — n.d. 3 197611MGRGSHHHHHHARSQVTWHHLADTVTTKNRKCTDSYIGWNXANAPKASAI — n.d. 4 197530MGRGSHHHHHHARSIIVIHNAIQTHTPHQVSIWCPPKHNRDANAPKASAI — n.d. 5 197557MGRGSHHHHHHARSSHCRHRNCHTITRGNMRIETPNNIRKDANAPKASAI ✓ ✓ 6 197596MGRGSHHHHHHARSTMNTNRMDIQRLMTNHVKRDSSPGSIDANAPKASAI ✓ ✓ 7 197552MGRGSHHHHHHARSLSALRRTERTWNTIHQGHHLEWYPPADANAPKASAI — n.d. 8 197541MGRGSHHHHHHARSWTSMQGETLWRTDRLATTKTSMSHPPDANAPKASAI — n.d. 9 197588MGRGSHHHHHHARSNVIPLNEVWYDTGWDRPHRSRLSIDDDANAPKASAI ✓ ✓ 10 197635MGPGSHHHHHHARSCLATRNGFV.MNTDRGTYVKRPTVLQDANAPKASAI ✓ — 11 197797MGRGSHHHHHHARSWGLSGTQTWKITKLATRLHHPEFETNDANAPKASAI — n.d. 12 197888MGRGSHHHHHHARSWRWHNWGLSDTVASHPDASNSLNMMYDANAPKASAN — n.d. 13 197897MGRGSHHHHHHLDLWGPPSGSPRTRSTTGTSTTSSPSTPGTLTLRRHPH — n.d 14 197825MGRGSHHHHHHARSWQPEVKMSSLVDTSQTVGAAVETRTTDANAPKASA ✓ — 15 198000MGRGSHHHHHHARSWRDTRKLHMRHYFPLAIDSYWDHTLRDANAPKASAI ✓ — 16 197983MGRGSHHHHHHHRSWTSMQGETLWRTDRLATTKTSMSHPPDANAPKASAI — n.d. 17 197998MGRGSHHHHHHHARSPLWYHYNCWDTICLADWLKDRPHGVYDANAPKASA — n.d. 18 197947MGRGSHHHHHHARSVGTTIRIAQDTEHYRNVYHKLSQYSRDANAPKASAI ✓ — 19 197954MGRGSHHHHHHARSVGTTIRIAQDTEHYRNVYHKLSQYSRDANAPKASAI — n.d. 20 197971MGRGSHHHHHHARSNVIPLNEVWYDTGWDRPHRSRLSIDDDANAPKASAI — n.d. *Test 1represents the target binding ability of free peptides (0) and test 2represents the binding specificity of variants in the presence of APprocess fluids (0). Variants that are positive in both assays are 5, 6and 9.

F. Peptide Production By Chemical Synthesis

Eight different peptides were produced by chemical synthesis in form ofN-terminal biotinylated peptides. The Biotin group was spaced via ashort hydrophilic linker (PEG2=8-Amino-3,6-dioxaoctanoic acid). Two ofthese 8 peptides (PP26-5 c and gurmarin-9 c) were additional synthesizedin form of C-terminal tagged biotinylated peptides (via an additionalC-terminal Lysine). The peptides were automatically synthesized usingthe Fmoc/But strategy according to Sheppard, purified by HPLC andsubsequently lyophilized. The quality of all purified peptides wasconfirmed by mass spectroscopy. The target quantity of each peptidesynthesis was 5 mg purified peptide. An overview about yield and purityof the synthetic peptides after purification is given in Table 5.

TABLE 5 Peptide Synthesis of Pertussis Toxin Binding Peptides* SelectionClone Seq # Sequence Purity (%) Yield (mg) pp26 5 c 197557RSSHCRHRNCHTITRGNMRIETPNNIRKDAK 90-95 7.7 pp26 5 n 197557RSSHCRHRNCHTITRGNMRIETPNNIRKDA 90-95 7.6 pp26 6 n 197596RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA 90-95 6.3 pp26 9 n 197588RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA 90-95 5.8 pp26 15 n  198000RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA 90-95 4.8 gurmarin 9 c 194259SGCVKKDELCARWDLVCCEPLECIYTSELYATCGK 70 1.0 gurmarin 9 n 194259SGCVKKDELCARWDLVCCEPLECIYTSELYATCG 80-90 4.0 gurmarin 10 n  194264SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG 90-95 4.9 gurmarin 15 n  194511SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS 90-95 6.3 gurmarin 19 n  194737SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG 90-95 6.7 *Abbreviation c in theclone name indicates C-terminal biotinylated peptides, abbreviation nindicates N-terminal biotinylated peptides.

All pp26 peptides were dissolved in 100 mM HEPES, pH 7.4, 200 mM NaClwith a final concentration of 100 μM. All gurmarin peptides weredissolved in 100 mM HEPES, pH 7.4, 200 mM NaCl, 2 mM GSH, 1 mM GSSG witha final concentration of 100 μM and subsequently incubated undernitrogen for at least 48 hours to allow structural folding.

G. Peptide Production By Bacterial Expression

The peptides which were identified as binders to the pertussis toxinwere subcloned in frame to glutathione-S-transferase (GST) and expressedbacterially. The GST-tag enhances the solubility and allows purificationusing Glutathione Sepharose. An engineered protease cleavage siterecognized by the specific PreScission™ protease allows removal of theGST-tag releasing the peptide. The PreScission™ protease itself is afusion protein of GST and human rhinovirus (HRV) type 14 3C protease andspecifically recognizes the sequence Leu-Phe-Gln*Gly-Pro cleavingbetween the Gln and Gly residues. After the cleavage the uncleavedproduct as well as the protease can be removed from the cleavagereactions using Glutathione Sepharose.

H. Construction of Expression Vectors 1. Construction of GST Fusions Forpp26-Variants

As template for PCR served the pCR2.1 vector containing the sequences ofthe identified pp26 binders to PT. The products obtained in a PCR usingthe oligonucleotides #467 (5′-CATGCCATGGGACGTGGCTCACATCATC-3′) and #468(5′-phosphate-GGGTTAAATAGCGGATGCCTTCGGAGCGTTAGCGTC-3′) with Pwo DNApolymerase (Roche) were digested with NcoI (New England Biolobs). Amodified vector (pGEX6P (Amersham/Pharmacia) containing an additionalNcoI site) was digested with NcoI/SmaI (New England Biolobs) and the PCRproduct was directionally cloned into the NcoI/SmaI site of this vector.After transformation in TOP10 (Invitrogen) positive clones wereidentified by colony PCR and verified by sequencing.

2. Construction of GST Fusions For Gurmarin-Variants

As template for PCR served the pCR2.1 vector containing the sequences ofthe identified gurmarin binders to PT. The products obtained in a PCRusing the oligonucleotides #464 (5′-GGAGATCTCATATGCACCATCACCATCACCATAGTGGC-3′) and #465(5′-phosphate-GGGTTAAATAGCGGATGCTACTAGGC-3′) with Pwo DNA polymerase(Roche) were digested with NdeI (New England Biolobs). A modified vector(pGEX6P (Amersham/Pharmacia) containing an additional NdeI site) wasdigested with NdeI/SmaI (New England Biolobs)and the PCR product wasdirectionally ligated into the NdeI/SmaI site of this vector. Aftertransformation in TOP10 (Invitrogen) positive clones were identified bycolony PCR and verified by sequencing (Table 6).

TABLE 6 Vectors used for bacterial expression Plasmid number pp26 pS840pGEX6P-(His)₆-pp26 K5 pS850 pGEX6P-(His)₆-pp26K6 pS841pGEX6P-(His)₆-pp26K9 pS842 pGEX6P-(His)₆-pp26K15 gurmarin pS836pGEX6P-(His)₆-gurmarin K9 pS837 pGEX6P-(His)₆-gurmarin K10 pS838pGEX6P-(His)₆-gurmarin K15 pS839 pGEX6P-(His)₆-gurmarin K19

3. Expression And Purification of GST-pp26 Fusions

The bacterial strain Rosetta (DE3) pLysS (Novagen) was transformed withplasmid DNA (see Table). The transfomands of the pp26 variants weregrown at 37° C. 250 rpm to an OD600 of ˜0.5 and induced by the additionof 1 mM IPTG for 4 h. In case of gurmarin-GST-fusions the induction wasperformed for 2.5 hours using 0.33 mM IPTG. After harvesting thebacterials, cells were resuspended in PBS-KMT (10 mM Na phosphate, pH7.5, 130 mM NaCl, 3 mM KCl, 1 mM MgCl, 0.1% Tween-20), containing 1 mM2-Mercaptoethanol, protease inhibitors and 1 mM Lysozyme, incubated for30 min at RT and disrupted by sonification. The soluble supernatantafter centrifugation was transferred to GSH sepharose column forpurification. After washing the column with 10 column volumes of 20 mMHepes, pH 7.5, 150 mM NaCl the GST fusion protein was eluted with 20 mMGSH and analyzed on a SDS gel to confirm expression.

4. Peptide Generation By Removal of GST PreScission™ Protease

An example for PreScission™ cleavage of one peptide from the GST-peptidefusion is shown below. The GST-tag was removed by incubation withPreScission™ Protease (Amersham Pharmacia): 2.5 mg of fusion protein wasincubated with 160U PreScission™ and digested for 16 hours at 5° C. onthe sealed GSTrap FF column containing the bound GST fusion protein.After the overnight incubation a second GSTrap FF column was connectedto remove the GST-tagged protease PreScission™. The sample was appliedwith a flow rate of 0.2 ml/min, the flow through was collected in smallaliquot samples and analyzed by SDS gel electrophoresis and the amountof peptide was calculated by OD₂₈₀ measurement (ca. 700 μg).

Example 3 Affinity Purification of PT A. Analysis of FermentationSupernatant On Denaturing Gels

Two process fluids were considered as potential starting material foraffinity chromatography process:

Sample A Concentrated culture filtrate containing 10-50 μg/ml(˜0.09-0.45 μM) crude PT, fermentation supernatant

Sample B Absorption chromatography supernatant containing 9-45 μg/ml(˜0.08-0.4 μM) crude PT

To visualize the complexity of these process fluids, both samples wereanalyzed by denaturating polyacrylamid gelelectrophoresis. Mainly highmolecular weight components of sample A are removed by the absorptionchromatography (sample B).

B. Immobilization of Synthetic Biotinylated Core Peptides ToStreptavidin Sepharose And Verification of Binding To Purified PT 1.Peptide Immobilization To Streptavidin Sepharose

For binding of biotinylated peptides to streptavidin sepharose (AmershamHigh Performance 71-5004-40), 200 μl of 50% slurry of pre-washedstreptavidin sepharose were incubated with 1 nmol peptide (10 μl of 100μM peptide solution) in 1 ml HEPES buffer (20 mM HEPES, pH 7.5, 150 mMNaCl, 0.025% TritonX-100) at 4° C. Under the applied conditions the highbinding capacity of streptavidin sepharose should allow immobilizationof 100% of the biotinylated peptide (10 pmol peptide per μl packedsepharose).

2. Binding of Purified PT To Immobilized Peptides

200 μl of sepharose loaded with peptides (10% ige slurry, containingimmobilized ˜200 pmol peptide) were transferred to a Mobicol column(MoBiTec, 10 μm filter) and the supernatant was removed bycentrifugation for 1 min at 2000 rpm. After 4 washes with HEPES buffer,the sepharose was resuspended in 200 μl HEPES buffer containing 100 pmolpurified Pertussis Toxin and incubated on a rotating wheel for 1 hour atroom temperature. The unbound fraction was separated by centrifugation(supernatant after binding; applied to gel analysis). Subsequently thepeptide-streptavidin sepharose was washed three-times with cold HEPESbuffer (each 200 μl) and resuspended in 20 μl loading buffer (30 mMTris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005%Bromphenol Blue) to elute bound PT. After 5 min incubation at 95° C. theloading buffer was collected by centrifugation and subsequently used forgel analysis (FIG. 15). As a control streptavidin sepharose withoutpeptide was contacted with PT under identical conditions. Under theapplied conditions the Pertussis toxin peptide binder clones pp26 5 n, 5c, 9 n, 15 n and the gurmarin clones 10 n, 19 n, 15 n, 9 n show a clearbinding to purified PT. All positive binder candidates were able to bindthe intact hexameric PT.

C. Immobilization of Synthetic Biotinylated Core Peptides ToStreptavidin Sepharose And Verification of Binding To PT Out of theFermentation Supernatant

Peptide immobilization to streptavidin sepharose and binding analysis toPT out of fermentation supernatants was performed as described inchapter 0 with the exception that the peptide streptavidin sepharoseswere incubated with 200 μl Sample A (fermentation supernatant) or with200 μl Sample B (absorption chromatography supernatant column, seechapter 0) and were subsequently washed 4-times with HEPES buffer at RT.The results of the binding analysis is presented in FIG. 16. Under theapplied conditions the Pertussis toxin pp26 binder clones 9 n, 15 n andthe gurmarin clones 9 n, 15 n were able to bind very efficiently theintact PT hexamer out of the fermentation supernatants Sample A andSample B. Note that under the applied conditions the pp26 binder clone 5and the gurmarin binder clones 10 and 19 might bind PT with loweraffinity. Although the PT binding to these peptides out of Sample A andSample B were not detected under the conditions, these binders might bestill qualified for application as ligand in an affinity chromatographycolumn (a column allows retention of PT by rebinding effects andtherefore would minimize the k_(off) problematic).

D. Thermodynamic Data On Immobilized Peptides

For estimation of peptides binding capacity 20 pmol ofsepharose-immobilized peptides were incubated with an excess of 100 pmolPT in a volume of 200 μl HEPES buffer (corresponds to 500 nM PT). Afterwashing, the fraction of peptide-streptavidin sepharose bound PT wasquantified by gel analysis. This allows directly to calculate thefraction of binding active peptide under the applied conditions(assuming the PT/peptide binding ratio is 1:1). Under the assumptionthat a concentration of 500 nM PT is high enough to reach B_(max) forall peptides. The results of the analysis are shown in Table 7. Thevalues presented therein are estimations for the expectable bindingcapacities of the peptides. An exact evaluation of binding capacity(B_(max)) and dissociation constant (K_(D)) of the most suitable bindermay also be performed.

TABLE 7 Overview about fraction of binding active peptides under theapplied experimental conditions Peptide pp26 pp26 pp26 gurm gurm gurmgurm name 5n 9n 15n 9n 10n 15n 19n Fraction >5% ¹ >50% >12.5% >12.5%|>5% ¹ >50% >5% ¹ of binding active peptide ¹ Calculation difficultbecause signals were near the detection limit

E. Analysis of the Stability of the Purified Pertussis Toxin HexamerUnder Defined Buffer Conditions (pH, Salt Concentration, Detergents),Using Acceptable Quality Grade Raw Materials Versus Health AuthoritiesRequirements

The Pertussis toxin hexamer stability was tested under a broad range ofpH and salt conditions on a BIAcore 2000 instrument. For this purpose2000 RU of biotinylated PT were loaded on a streptavidin chip.Subsequently different buffers were applied to the chip immobilized PTfor 2 min with a flow rate of 30 μl/min. After the end of each bufferinjection the chip was equilibrated with HBS/EP running buffer (0.01 MHEPES pH 7.4, 0.15 M NaCl 3 mM EDTA 0.005% polysorbate 20 (v/v), atleast 2 min with a flowrate of 30 μl/min). The difference of themeasured RU signal before and after buffer injection correlates to thereduction of PT hexamer on the chip. This reduction was interpreted asloss of stability of PT hexamer under the applied buffer conditions.

The analyzed pH range was between pH 2 and 10.5 using the followingbuffers: 10 mM glycin buffer (BIAcore, pH 2; 2.5; 3), 10 mM acetatebuffer (BIAcore, pH 4; 4.5; 5; 5.5), 50 mM Tris/HCl (pH 8.5) and 100 mMcarbonate buffer (pH 9.6 and 10.5). BIAcore sensograms demonstrating theinfluence of the pH on the PT hexamer stability were generated, and theresults of the BIAcore analysis are summarized in Table 8. Under theapplied conditions, Pertussis toxin hexamer was shown to be stable overa broad pH range between pH 2.5-10.5.

TABLE 8 Pertussis toxin hexamer stability under different pH conditions.pH 2 2.5 3 4 4.5 5 5.5 8.5 9.6 10.5 PT hexamer 93 98 100 100 100 100 100100 98 95 stability (%)

The influence of different salt conditions on the PT hexamer stabilitywere investigated in comparable experiments on the BIAcore 2000instrument for NaCl, KCl and MgCl₂ at pH 5.0 (10 mM acetate buffer) andpH 8.5 (50 mM Tris/HCl) respectively. An overview about PT hexamerstability under the applied salt conditions is shown in Table 9. Thehexamer was stable in buffer (at pH 5 and 8.5) containing up to 2.5 MNaCl or up to 2 M KCl. In case of MgCl₂ the PT hexamer was stable in abuffer containing up to 2 M MgCl₂ at pH 8.5.

TABLE 9 Pertussis toxin hexamer stability under different saltconditions PT hexamer stability pH 5 pH 8.5 NaCl 0-2.5 M 0-2.5 M stablestable KCl 0-2.0 M 0-2.0 M stable stable MgCl₂ Nd 0-2.0 M stable

F. Establish Defined Wash And Elution Conditions Allowing A SpecificAffinity Purification of PT Out of Fermentation Supernatant (pH, SaltConcentration, Detergents)

After the determination under which conditions the Pertussis toxinhexamer is stable, the next step was to investigate the wash and elutionconditions for the bound Pertussis toxin to the immobilized peptidespp26 clone 9 and 15 and gurmarin clone 9 and 15.

1. Evaluation of PT/Peptide Stability Using the BIAcore 2000 Instrument

The stability of PT/peptide complexes were investigated using theBIAcore 2000 instrument under different pH and salt conditions that wereshown before not to interfere with the PT hexamer stability. 500-1000 RUof the synthetic peptides were immobilized on BIAcore streptavidinchips. To allow binding of PT to the immobilized peptides, 20 nMpurified PT in HEPES buffer was injected for 1 minute. Afterequilibration with HBS/EP running buffer (0.01 M HEPES pH 7.4, 0.15 MNaCl 3 mM EDTA 0.005% polysorbate 20 (v/v)) the PT/peptide complexeswere washed by injection of

(a) 100 mM carbonate buffer at pH 10.5 and 9.5

(b) 10 mM acetate buffer at pH 5.5, 5.0, 4.5, and 4.0

(c) 10 mM glycin buffer at pH 3.0 and 2.5

(d) 0.5, 1.0, 1.5, 2.0 M NaCl in 10 mM acetate buffer buffer, pH 6.0,

(e) 0.5, 1.0, 1.5, 2.0 M NaCl in 50 mM Tris/HCl buffer, pH 8.5,

(f) 0.5, 1.0, 1.5, 2.0 M KCl in 10 mM acetate buffer, pH 6.0,

(g) 0.5, 1.0, 1.5, 2.0 M KCl in 50 mM Tris/HCl buffer, pH 8.5,

(h) 0.5, 1.0, 1.5, 2.0 M NaCl in 10 mM acetate buffer, pH 6.0,

(i) 0.5, 1.0, 1.5, 2.0 M NaCl in 50 mM Tris/HCl buffer, pH 8.5.

After the end of each buffer injection, the chip was equilibrated withHBS/EP running buffer. The loss of PT hexamer on the chip under theapplied buffer conditions (difference of measured RU signal before andafter buffer injection) reflects the PT/peptide complex stability. Anoverview about the pH range stability and salt stability of allPT/peptide complexes is summarised in Table 10. All of the PT/peptidecomplexes were completely destabilized in the presence of 100 mMcarbonate, pH 10.5 as well as 10 mM glycin, pH 2.5. For gurmarin peptide9, buffers containing 2.5 M NaCl or at least 0.5 M MgCl₂ interfere withPT/peptide complex stability. PT complexes with gurmarin peptide 15 wereadditionally destabilized in the presence of at least 1.5 M MgCl₂ in 50mM Tris/HCl, pH 8.5).

TABLE 10 Effect of different pH and salt conditions on the stability ofthe PT/peptide complexes pp26 peptide 9 pp26 peptide 15 gurmarin peptide9 gurmarin peptide 15 3-9 3-9 3-9 3-9 stable, stable, stable, stable, pHrange instable at pH 2.5 or instable at pH 2.5 or instable at pH 2.5 orinstable at pH 2.5 or stability of 10.5 10.5 10.5 10.5 the complex pH 6pH 8.5 pH 6 pH 8.5 pH 8.5 pH 6 pH 8.5 NaCl 2 M 2 M 2 M 2 M strongsensitive to salt sensitive to salt stability of stable stable stablestable the complex KCl 2 M 2 M 2 M 2 M strong sensitive to saltsensitive to salt stability of stable stable stable stable the complexMgCl₂ stable Sensitive stable up to Elution ≧ 1.5, Elution ≧ 0.5 MElution ≧ 1 M, Elution ≧ 1 M, stability of from 125 mM, 2 M but notcomplete complete the complex complete complete elution ≧ elution ≧elution ≧ 2 M 2 M 2 M

2. Evaluation of Wash Conditions For Purification of PT On PeptideStreptavidin Sepharose

Wash conditions were tried to apply close to the established conditionsfor pertussis toxin purification process on asialofetuin (washing with50 mM Tris/HCl, pH 7.5, with or without 1 M NaCl). The Pertussis toxinpurification protocol was optimized for the peptides pp26 clone 9 and 15and gurmarin clone 9 and 15. 200 pmol of each peptide immobilized on 20μl sepharose were incubated with 100 μl 50 mM Tris/HCl, pH 7.5 and 100μl sample A or sample B to allow binding of PT. Subsequently the peptidesepharose with bound PT fraction was washed under 3 differentconditions, as shown below:

(a) 3 times with 200 μl 50 mM Tris/HCl, pH 7.5;

(b) 3 times with 200 μl 50 mM acetate pH 6.0; and,

(c) 6 times with 200 μl 50 mM acetate pH 6.0.

After washing remaining material was eluted from the sepharose with 20μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol,12.5% Glycerol, 0.005% Bromphenol Blue). All elutions were subsequentlyanalyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silverstaining (FIG. 17).

Washing with 50 mM acetate, pH 6.0, is more stringent and reduces theback ground of high molecular weight impurities more efficient thanwashing with 50 mM Tris/HCl, pH 7.5. But under these washing conditionsthe PT/peptide complexes are less stable, especially in case of thegurmarin peptide 9 and repeated washes with 50 mM acetate, pH 6.0 (6washes). In contrast to 50 mM Tris/HCl, pH 7.5, the loss of peptideimmobilized PT was more dramatic when washing with 50 mM acetate, pH 6.0was repeated 10 to 20 times (as an example shown for pp26 peptide 9 inFIG. 18).

3. Evaluation of Elution Conditions For Purification of PT On PeptideStreptavidin Sepharose

Elution of PT from peptide sepharose was tested under conditions thatare compatible with hexamer stability.

a. Elution By MgCl₂

As shown above by BIAcore 2000 measurements all PT/peptide complexeswere sensitive against 2 M MgCl₂, conditions that were shown not to becritical for PT hexamer stability. The elution efficiencies of definedMgCl₂ concentrations were evaluated for PT that was bound onstreptavidin sepharose via one of the four immobilized syntheticpeptides. 400 pmol of each peptide immobilized on 20 μl sepharose wereincubated with 100 μl 50 mM Tris/HCl, pH 7.5 and 100 μl sample A toallow binding of PT. After 4 washes with 50 mM Tris/HCl, pH 7.5 (200 μleach), the bound fraction of PT was eluted using 3 consecutive 20 μlvolumes of

(a) 0.2 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

(b) 0.5 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

(c) 1.0 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

(d) 1.5 M MgCl₂ in 50 mM Tris/HCl, pH 8.5, or

(e) 2.0 M MgCl₂ in 50 mM Tris/HCl, pH 8.5.

Remaining material was afterwards eluted from the peptide streptavidinsepharose with 20 μl loading buffer (30 mM Tris, pH 6.8, 1% SDS, 1%β-Mercaptoethanol, 12.5% Glycerol, 0.005% Bromphenol Blue). All elutionswere analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) andsilver staining (FIG. 19). As shown in the experiment elution with MgCl₂was more efficient for the gurmarin peptides than for the pp26 peptidesalthough a substantial amount of PT still remained on the peptidestreptavidin sepharose.

b. Elution By pH-Shift

The BIAcore measurements revealed that PT was elutable from all peptideswith acidic (pH of 2.5) or basic (pH of 10.5) buffer conditions thatwere not critical for PT hexamer stability (50 mM glycin, pH 2.5 moregentle for PT hexamer stability than 100 mM carbonat buffer, pH 10.5,see xxx). 200 pmol of each peptide immobilized on 20 μl sepharose wereincubated with 100 μl 50 mM Tris/HCl, pH 7.5 and 100 μl sample A toallow binding of PT. After 4 washes with 50 mM Tris/HCl, pH 7.5 (200 μleach), PT was eluted from the peptide streptavidin sepharose by 3consecutive 40 μl elutions with 50 mM glycin, pH 2.5, or 100 mMcarbonate buffer, pH 10.5. Remaining material was subsequently elutedfrom the peptide streptavidin sepharose with 20 μl loading buffer (30 mMTris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5% Glycerol, 0.005%Bromphenol Blue). All elutions were analyzed by PAGE on 12%Bis-Tris-Gels (MES running buffer) and silver staining (FIG. 20). Nearlyall of PT was elutable from the peptide streptavidin sepharose using 50mM glycine, pH 2.5 as well as using 100 mM carbonate buffer, pH 10.5.

4. Apply Optimized Conditions For Small Scale Purification Scheme,Confirm Binding Capacity a. Purification of PT From Sample B UnderOptimized Wash And Elution Conditions (4 μl Column)

Optimized wash and elution conditions were combined to allow thepurification of PT on peptide streptavidin sepharoses out of Sample B.To reduce unspecific binding of PT the optimal peptide/streptavidinsepharose ratio was titrated for each peptide before. Subsequently theSample B/peptide streptavidin sepharose ratio was optimized in respectto high recovery of PT per expectable high (moderate) input of peptide.These conditions were applied to the following small scale columnpurifications.

For pp26 peptide 9 and gurmarin peptide 15, the immobilization tostreptavidin sepharose was performed by incubation of 16 μl streptavidinsepharose with 1600 pmol peptide. In case of pp26 peptide 15, 16 μlstreptavidin sepharose was incubated with 6000 pmol peptide (pp26/15binds with lower efficiency to the streptavidin sepharose, might beexplainable by incomplete peptide biotinylation). For gurmarin peptide9, 8000 pmol were immobilized on 80 μl streptavidin sepharose.Subsequently the washed peptide streptavidin sepharoses were equallysubdivided and transferred to 4 Mobilcom columns (with 10 μM filters).

Each column (containing 4 μl sepharose with 400 pmol peptide for pp26/9and gur/15; 4 μl with undefined amount bound peptide pp26/15; 20 μl with2000 pmol peptide for gur/9) was incubated with 400 μl Sample B(adjusted to pH 7.0-7.5 by addition of HCl) to allow binding of PT.After 5 washes with 50 mM Tris/HCl, pH 7.5 (each 100 μl), PT was elutedfrom the peptide streptavidin sepharose by consecutive elutions (3elutions for pp26/9 and gur/15; 4 elutions for pp26/15 and gur/9), asfollows:

(a) with 50 mM glycine, pH 2.5 (each 20 μl) in case of column 1, or

(b) with 100 mM carbonate buffer, pH 10.5 (each 20 μl) in case of column2, or

(c) with 2 M MgCl₂ in 50 mM Tris, pH 8.5 (each 20 μl) in case of column3.

Remaining material on column 1-3 as well on column 4 was subsequentlyeluted from the peptide streptavidin sepharoses by elution with 20 μlloading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5%Glycerol, 0.005% Bromphenol Blue). All elutions were analyzed by PAGE on12% Bis-Tris-Gels (MES running buffer) and silver staining (FIGS. 21,22). To calculate the yield of PT after purification on the peptidestreptavidin sepharoses the pooled elutions 1-3 were analyzed by PAGEand silver staining and compared to defined amounts of purified PTseparated on the same gel allowing an estimation (FIGS. 21B and 22B).Based on the gel estimation, the yield of purified PT was calculated asshown in Table 11.

TABLE 11 Calculation of the Pertussis Toxin yield after small scalecolumn purification from sample B with pp26 peptide 9 or gurmarinpeptide 15 as affinity ligands Total Yield relative to Yield relative tothe Estimation from yield of input PT in amount of sepharose PeptideElution with FIG. 13B and 14B PT sample B* bound peptide pp26 peptide 9Glycin pH 2.5 2 pmol PT in 3/120 80 pmol >48% 20% of pooled elutionsCarbonat pH 10.5 elution 2 pmol PT in 3/120 80 pmol >48% 20% of pooledelutions MgCl₂ 1 pmol PT in 6/120 20 pmol >12%  5% of pooled elutionsgurmarin peptide 15 Glycin pH 2.5 2 pmol PT in 3/120 80 pmol >48% 20% ofpooled elutions Carbonat pH 10.5 elution 2 pmol PT in 3/120 80 pmol >48%20% of pooled elutions MgCl₂ 1 pmol PT in 3/120 40 pmol >24% 10% ofpooled elutions *according to the documentation related to PT, theexpected PT concentration of sample B is 9-45 μg/ml, corresponding to0.8-0.41 pmol/μl. Calculation was performed as following: 400 μl ofsample B × 0.41 pmol/μl = 164 pmol input PT

b. Determination of PT Yield During Affinity Purification Using VaryingPeptide Densities On Streptavidin Sepharose

The PT binding to peptide streptavidin sepharose was investigated independence of varying concentration of peptide immobilized on thestreptavidin sepharose as affinity ligand. For immobilization 1 μlvolume of streptavidin sepharose was incubated with increasing amountsof peptide pp26/9 or gurmarin/15 (100, 200, 300, 400, 500, 1000 pmolpeptide). Unbound fractions of peptides were removed from the sepharoseby 3 washes with 50 mM Tris/HCl, pH 7.5 (on column). Subsequently eachpeptide streptavidin matrix was incubated with 600 μl Sample A to allowbinding of PT. After 80 min each matrix was washed four times with 50 mMTris/HCl, pH 7.5 (200 μl each) and subsequently eluted with 20 μl gelloading buffer (30 mM Tris, pH 6.8, 1% SDS, 1% β-Mercaptoethanol, 12.5%glycerol, 0.005% Bromphenol blue; incubation for 10 min at 95° C.).Elutions were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer)and silver staining (FIG. 23). Amount of PT that was bound to peptidestreptavidin sepharose was calculated by densitometric evaluation andplotted as a function of the amount of peptide initially used forimmobilization to streptavidin sepharose (shown for pp26/9 in FIG. 23).A maximum of PT binding was reached when 300-400 pmol peptide were usedfor immobilization to 1 μl streptavidin sepharose. Higher amounts ofpeptide did not result in higher PT binding probably reflecting effectsof steric hindrance of PT.

The effectively bound fraction of peptide (pp26/9 or gurmarin/15) whenan input of 400 pmol peptide was used for immobilization to 1 μlstreptavidin sepharose, was evaluated by PAGE on a 12% Bis-Tris-Gel (MESrunning buffer) and silver staining after elution with gel loadingbuffer (heating at 95° C. for 10 min). Amount of elutable peptide wasestimated by direct comparison to defined amounts of purified PT on thesame gel (data not shown): for pp26/9: 100-150 pmol; for gurmarin/15: 50pmol.

C. Determination of PT Yield Using Varying Amounts Of Sample B AtConstant Concentration of Peptide Sepharose During Affinity Purification

For peptide immobilization 400 pmol pp26/9 or gurmarin/15 were incubatedwith 1 μl streptavidin sepharose for 1 h at RT. The peptide sepharosewas washed 3 times with 200 μl 50 mM Tris pH 7.5 buffer and subsequentlyincubated with varying amounts of Sample B (50, 66, 100, 200, 400, 600μl, adjusted before to pH 7.0-7.5 by addition of HCl) for 1 hour at RT.The affinity matrices were washed 4 times with 100 μl 50 mM Tris/HCl, pH7.5, and eluted by 4 consecutive elutions with 100 mM Carbonate bufferat pH 10.5 (each 20 μl). 5 μl of the pooled elutions (total 80 μl) wereanalyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer) and silverstaining. The amount of eluted PT was calculated on the basis of directcomparison to defined amounts of purified PT on the same gel as massstandard (FIG. 24, Table 12).

TABLE 12 Yield of PT Input PT Ratio Amount of PT relative to input(pmol) peptide:PT bound (pmol) amount of PT Input peptide 16K9 (pmol)100 300 1:3 ~100 33% 100 200 1:2 ~88 44% 100 100 1:1 ~40 40% 100 50 2:1~24 48% 100 33.3 3:1 ~16 48% 100 25 4:1 ~24 96% Input peptide 17K15(pmol) 100 300 1:3 ~80 27% 100 200 1:2 ~64 32% 100 100 1:1 ~56 56% 10050 2:1 ~16 32% 100 33.3 3:1 ~16 32% 100 25 4:1 ~8 32% Input asiaolfetuin(pmol) 100 200 1:2 ~8  4% 100 100 1:1 ~16 16% 100 85.6 20:17 ~8  9% 10050 2:1 ~8 16% 100 33.3 3:1 ~8 24% 100 25 4:1 ~8 35%

To compare the purification efficiencies of the peptide streptavidinsepharoses with asialofetuin sepharose a titration experiment withasialofetuin sepharose was performed in parallel under comparableconditions (same amount of affinity ligand per reaction immobilized onsepharose, corresponding to ˜100 pmol affinity ligand effectively boundto the sepharose). This was accomplished by incubation of 6.85 μl ofasialofetuin sepharose (batch number FA 053198: density 1.1 mg/ml, 14.6pmol/μl) with varying amounts of Sample B (50, 66, 100, 171.3, 200, 400μl, adjusted before to pH 7.0-7.5 by addition of HCl) for 1 hour at RT.Subsequently the asialofetuin sepharose was washed and bound PT waseluted and analyzed as described above. The binding efficiency ofpeptide streptavidin sepharose under the applied purification conditionswas significantly higher than the binding efficiency of asialofetuinsepharose.

d. Reutilization of Peptide Sepharose For Repeated PT binding andelution

To investigate the reusability of peptide loaded sepharose (pp26/9 andgurmarin/15) for repeated binding and elution of PT the sepharoses wereapplied for repeated cycles of PT binding, elution and regeneration (intotal 4 times). For peptide immobilization 600 pmol pp26/9 orgurmarin/15 were incubated with 2 μl streptavidin sepharose over nightat RT and subsequently washed 3 times with HEPES buffer. For binding ofPT each peptide streptavidin sepharose was incubated with 400 μl sampleB (adjusted to pH 7.0 -7.5 by addition of HCl) for 1 hour at RT andwashed 4 times with 50 mM Tris/HCl, pH 7.5 (each 200 μl). PT was elutedby 4 consecutive elutions with 100 mM Carbonate buffer at pH 10.5 (each20 μL). Subsequently the column matrices were regenerated by threewashes with 10 mM HCl (1×20 μl, 2×100 μL) and afterwards neutralized bytwo washes with 200 μl 50 mM Tris/HCl, pH 7.5. This binding, elution andregeneration procedure was applied to the peptide sepharose for threeadditional times. 4 μl of the pooled elutions (in total 80 μl) and 7 μlof the first regeneration buffer from each binding/elution/regenerationcycle were analyzed by PAGE on 12% Bis-Tris-Gels (MES running buffer)and silver stained, indicating that the peptide sepharose may bere-utilized. (FIG. 25).

5. Large-Scale FPLC-Purification of PT

Optimized conditions for PT binding and elution were applied for largescale FPLC purification (0.5 ml column), as shown below:

A) Immobilization of biotinylated peptide to streptavidin-sepharose: 200nmol peptide pp26/9 were incubated for 1 h 30 min at room temperature ona rotating wheel with 1 ml 50% Streptavidin-sepharose in volume of 10 ml(HEPES-buffer). After incubation the sepharose was washed 3× with 50 mMTris pH 7.5.

B) Binding of PT (out of sample B): The estimated amount of peptideeffectively immobilized on 500 μl sepharose was 50 nmol. Thepeptide-sepharose was incubated with 25 ml sample B for 1 h 30 min atroom temperature in a head over tail rotator (assumed concentration ofPT ˜0.5 pmol/μl, corresponding to 12.5 nmol in 25 ml, corresponding to aratio of immobilized peptide to amount of PT of 4:1).

C) FPLC-column: After incubation the sepharose was transferred to acolumn (Pharmacia HR 5/5) During packing of the column the sepharose waswashed with 50 mM Tris pH 7.5 (2-3 ml). Subsequently the column wastaken in the flow path and washed with 20 column volumes (10 ml) 50 mMTris ph 7.5. Immobilized PT was eluted with 11 ml 100 mM carbonatebuffer pH 10.5. The elution fractions were collected in 500 μl fractions(Pharmacia Fraction Collector FRAC-100) and the elution profile wasevaluated by measurement of the UV absorbance at 280 nm. After elutionthe column was washed with 1.5 ml 50 mM Tris pH 7.5 and subsequentlyregenerated with 2.5 ml 10 mM HCl followed by neutralization with 10 ml50 mM Tris pH 7.5.

D) analysis of elution fractions and calculation of yield: The elutionfractions were analyzed by PAGE (12% Bis-Tris-Gel, MES running buffer)and silver staining (FIG. 26). Concentration of PT was determined bymeasuring the absorbance of the elution fractions at 280 nm (A₂₈₀) andcomparing these results with a calibration curve prepared with purifiedPT (see table in FIG. 26).

The amount of PT was additionally calculated on the basis of directcomparison to defined amounts of purified PT on the same gel as massstandard. Gel estimation leads to a yield of 8100 pmol PT. Thiscorrelates very well with the concentration determination using A₂₈₀. Ifit is assumed that 25 ml sample B contains 1125 μg of PT, more than69%-72% is eluted of PT under these conditions. This result was verifiedby repetition of the FPLC run using the same peptide-sepharose afterregeneration to-bind PT out of 25 ml sample B. In this experiment, 803μg PT was purified (A₂₈₀) (Table 13).

TABLE 13 Determination of concentration of PT in elution fraction (FPLCrun #2) using A₂₈₀ A₂₈₀ μg/ml Elu1 0 0 Elu2 0 0 Elu3 0.091 85 Elu40.4185 391 Elu5 0.354 331 Elu6 0.2835 265 Elu7 0.212 198 Elu8 0.148 138Elu9 0.0975 91 Elu10 0.0585 55 Elu11 0.0315 29 Elu12 0.025 23 Total 3-12=803 μg

TABLE 14 Summary of PT Purification Results Relative Yield versus YieldPT in 12x input amount of PT 0.5 ml (1125 μg in 25 ml) fractions (6 ml)(pmol/pmol or μg/μg) Purity 1. purification run 772-813 μg 69%-72%Comparable to PT purified on asialofetuin sepharose, 100% 2.purification run   803 μg 71% Comparable to PT purified on asialofetuinsepharose, 100%

6. Evaluation Of Equilibrium And Rate Constants of the pp26 Peptide9/Pertussis Toxin Complex Formation

Equilibrium constants and rate constants for the pp26 K9/PT complexformation were evaluated using the BIAcore 2000 instrument in HBS/EPrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%(v/v) polysorbate 20) at room temperature. Binding of varyingconcentrations of pp26-K9 (concentrations between 2.5 nM and 100 nM) toPT immobilized on a CM5 chip (immobilization of 6000 RU via aminecoupling method) were analyzed at a flow rate of 30 μl/min. Quantitativeelution of PT bound peptides were obtained by using 3 mM HCl, pH 2.5.Deducible equilibrium and rate constants were analyzed using theBIAevaluation software, the results of which are shown below:

Dissociation equilibrium constant K_(D)→ 7.5 × 10⁻⁹ M Associationequilibrium constant K_(A)→ 1.3 × 10⁻⁸ M⁻¹ Association rate constantk_(on) → 1.3 × 10⁵ M⁻¹ × s⁻¹ Dissociation rate constant k_(off) → 10⁻³s⁻¹

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1-51. (canceled)
 52. A peptide having the ability to bind pertussistoxin, the peptide being selected from the group consisting of:RSSHCRHRNCHTITRGNMRIETPNNIRKDA (pp26-5); RSTMNTNRMDIQRLMTNHVKRDSSPGSIDA(pp26-6); RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9);RSWRDTRKLHMRHYFPLAIDSYWDHTLRDA (pp26-15);SGCVKKDELCARWDLVCCEPLECIYTSELYATCG (G-9);SGCVKKDELCELAVDECCEPLECFQMGHGFKRCG (G-10);SGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15);SGCVKKDELCELAIDECCEPLECTKGDLGFRKCG (G-19); NVIPLNEVWYDTGWDRPHRSRLSIDDD,VGTTIRIAQDTEHYRNVYHKLSQYSR, WTSMQGETLWRTDRLATTKTSMSHPP,LSALRRTERTWNTIHQGHHLEWYPPA, LSRLATTERTWDRIHQGHHLEWHPPA,TMNTNRMDIQRLMTNHVKRDSSPGSI, LSALMRTERTWNTIHQGHHLEWYPPA,CLATRNGFVMNTDRGTYVKRPTVLQ, and CLATRNGFVQMNTDRGTYVKRPTVLQ.


53. A peptide having the ability to bind pertussis toxin and the aminoacid sequence RSNVIPLNEVWYDTGWDRPHRSRLSIDDDA (pp26-9).
 54. A peptidehaving the ability to bind pertussis toxin and the amino acid sequenceSGCVKKDELCSQSVPMCCEPLECKWFNENYGICGS (G-15).
 55. A peptide of claim 52wherein at least one amino acid is conservatively substituted.
 56. Apeptide of claim 53 wherein at least one amino acid is conservativelysubstituted.
 57. A peptide of claim 54 wherein at least one amino acidis conservatively substituted.
 58. A peptide having the ability to bindpertussis toxin and comprising the amino acid sequence selected from thegroup consisting of: XXAXRXXXXXXNTXXXXXXXXXT, XXAXRXXXXXXNTXXXXXXXXXY,and VXXXXXXXXDTXXXXRXXXXXLS, where X is any amino acid.
 59. A peptidehaving the ability to bind pertussis toxin and comprising the amino acidsequence LGHGLGYAY.
 60. A peptide of claim 59 further comprising theamino acid sequence ELAVD, ELAID, or ARWDLV.
 61. A peptide having theability to bind pertussis toxin and comprising at least one of the aminoacid sequences TTASKS or KWTNEHFGT.
 62. A peptide of claim 61 comprisingthe amino acid sequences TTASKS and KWTNEHFGT.
 63. A method forgenerating a DNA-peptide fusion, said method comprising: (a) covalentlybonding a nucleic acid reverse-transcription primer to an RNA encoding apeptide, said reverse-transcription primer being bound to a peptideacceptor; (b) translating said RNA to produce the peptide, the peptidebeing covalently bound to the reverse-transcription primer; and, (c)reverse transcribing said RNA to produce a DNA-peptide fusion; whereinthe peptide of the DNA-peptide fusion has binding affinity for pertussistoxin.
 64. A method for generating a DNA-peptide fusion, said methodcomprising: (a) generating an RNA-peptide fusion; (b) hybridizing anucleic acid reverse-transcription primer to said fusion; (c) covalentlybonding said primer to said fusion; and (d) reverse transcribing the RNAof said RNA-peptide fusion to produce a DNA-peptide fusion; wherein thepeptide of the DNA-peptide fusion has binding affinity for pertussistoxin.
 65. A method for generating a DNA-peptide fusion comprising: (a)providing an RNA molecule covalently bonded to a peptide acceptor; (b)covalently bonding a nucleic acid reverse-transcription primer to themolecule of step (a); (c) translating said RNA molecule to produce apeptide, and (d) reverse transcribing said RNA molecule to produce aDNA-peptide fusion; wherein the peptide of the DNA-peptide fusion hasbinding affinity for pertussis toxin.
 66. A method for isolating aDNA-peptide fusion in which the peptide has binding affinity forpertussis toxin comprising the steps of, in combination: (a) covalentlybonding a nucleic acid reverse-transcription primer to an RNA encoding apeptide, said reverse-transcription primer being bound to a peptideacceptor; (b) translating the RNA to produce the peptide, the peptidebeing covalently bound to the reverse-transcription primer; and, (c)reverse transcribing the RNA to produce a DNA-peptide fusion; (d)contacting the DNA-peptide fusion with pertussis toxin bound to a solidsupport to form a DNA-peptide fusion-pertussis toxin complex; (e)isolating the complex from DNA-peptide fusions that did not complex withpertussis toxin; and, (f) isolating the DNA-peptide fusion from theDNA-peptide fusion-pertussis toxin complex.
 67. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 52 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 68. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 53 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 69. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 54 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 70. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 55 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 71. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 56 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 72. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 57 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 73. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 58 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 74. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 59 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 75. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 60 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 76. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 61 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 77. A method for purifyingpertussis toxin comprising contacting a biological solution containingpertussis toxin with a peptide of claim 62 bound to a solid support toform a pertussis toxin-peptide complex and isolating the complex fromother components in the biological solution.
 78. A DNA-peptide fusionprepared using the method of claim
 63. 79. A DNA-peptide fusion preparedusing the method of claim
 64. 80. A DNA-peptide fusion prepared usingthe method of claim
 65. 81. A DNA-peptide fusion prepared using themethod of claim 66.